U.S. patent application number 17/047013 was filed with the patent office on 2021-03-18 for non-invasive detection of response to a targeted therapy.
The applicant listed for this patent is The Johns Hopkins University. Invention is credited to Hatim Husain, Alessandro Leal, Jillian A. Phallen, Victor E. Velculescu.
Application Number | 20210079384 17/047013 |
Document ID | / |
Family ID | 1000005277667 |
Filed Date | 2021-03-18 |
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United States Patent
Application |
20210079384 |
Kind Code |
A1 |
Velculescu; Victor E. ; et
al. |
March 18, 2021 |
NON-INVASIVE DETECTION OF RESPONSE TO A TARGETED THERAPY
Abstract
Provided herein are method of determining the efficacy of
targeted therapy in a subject by detecting changes in levels of
cell-free tumor load (cfTL). In some aspects, the efficacy of
targeted therapy is determined a very short time after the targeted
therapy is administered. Also provided herein are method of
determining resistance to a targeted therapy in a subject by
detecting changes in levels of cell-free tumor load (cfTL).
Inventors: |
Velculescu; Victor E.;
(Dayton, MD) ; Phallen; Jillian A.; (Baltimore,
MD) ; Leal; Alessandro; (Baltimore, MD) ;
Husain; Hatim; (Baltimore, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Johns Hopkins University |
Baltimore |
MD |
US |
|
|
Family ID: |
1000005277667 |
Appl. No.: |
17/047013 |
Filed: |
April 12, 2019 |
PCT Filed: |
April 12, 2019 |
PCT NO: |
PCT/US2019/027207 |
371 Date: |
October 12, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62657618 |
Apr 13, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/1072 20130101;
C12N 15/1065 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0001] This invention was made with government support under grant
numbers NIH R01 grant (CA121113) awarded by the National Institutes
of Health. The government has certain rights in the invention.
Claims
1. A method of determining the efficacy of a targeted therapy in a
subject having cancer, comprising: detecting a first cell-free
tumor load (cfTL) in a biological sample isolated from the subject
at a first time point; detecting a second cfTL in a biological
sample obtained from the subject at a second time point, wherein
the subject has received at least one dose of the targeted therapy
between the first time point and the second time point; and
identifying the targeted therapy as being effective in the subject
when the subject exhibits a second cfTL that is reduced as compared
to the first cfTL.
2. The method of claim 1, wherein: detecting the first cfTL
comprises detecting a first level of the at least one genetic
alteration in circulating tumor DNA (ctDNA) in the biological
sample isolated from the subject at the first time point, wherein
the first cfTL corresponds to the first level of the at least one
genetic alteration; and detecting the second cfTL comprises
detecting a second level of the at least one genetic alteration in
circulating tumor DNA (ctDNA) in the biological sample isolated
from the subject at the second time point, wherein the second cfTL
corresponds to the second level of the at least one genetic
alteration.
3. (canceled)
4. The method of claim 3, wherein detecting the first level of the
at least one genetic alteration, detecting the second level of the
at least one genetic alteration, or both comprises: extracting
cell-free DNA from blood; ligating a low complexity pool of dual
index barcode adapters to the cell-free DNA to generate a plurality
of barcode adapter-ligated cell-free DNA segments; capturing the
plurality of barcode adapter-ligated cell-free DNA segments;
sequencing the plurality of captured barcode adapter-ligated
cell-free DNA segments; aligning the sequenced plurality of
captured barcode adapter-ligated cell-free DNA segments to a
reference genome; and identifying sequence alterations using
aligned sequences of multiple distinct molecules containing
identical redundant changes.
5. The method of claim 2, wherein the at least one genetic is a
mutation is in an EGFR gene, an ERBB2 gene, or both.
6. The method of claim 2, wherein the at least one genetic
alteration is a T790M mutation in the EGFR gene.
7. The method of claim 2, wherein the second level of the at least
one genetic alteration of ctDNA is at least about 90% lower than
the first level of the at least one genetic alteration of
ctDNA.
8. The method of claim 1, wherein: detecting the first cfTL
comprises detecting a first level of aneuploidy in the biological
sample isolated from the subject at the first time point, wherein
the first cfTL corresponds to the first level of aneuploidy; and
detecting the second cfTL comprises detecting a second level of
aneuploidy in the biological sample isolated from the subject at
the second time point, wherein the second cfTL corresponds to the
second level of aneuploidy.
9. The method of claim 8, wherein detecting the first level of
aneuploidy, detecting the second level of aneuploidy, or both
comprises: performing digital karyotyping, next generation
sequencing, array-based methods, and combinations thereof.
10. The method of claim 8, comprising: a) extracting a first sample
of cell-free DNA from blood at the first time point; ligating a low
complexity pool of dual index barcode adapters to the first
cell-free DNA sample to generate a first plurality of barcode
adapter-ligated cell-free DNA segments; capturing the first
plurality of barcode adapter-ligated cell-free DNA segments;
eluting the non-captured cell-free DNA to generate a first
non-captured cell-free DNA sample; detecting the first level of
aneuploidy in the first non-captured non-capture cell-free DNA; and
b) extracting a second sample of cell-free DNA from blood at the
second time point; ligating a low complexity pool of dual index
barcode adapters to the cell-free DNA to generate a plurality of
barcode adapter-ligated cell-free DNA segments; capturing the
plurality of barcode adapter-ligated cell-free DNA segments;
eluting the non-captured cell-free DNA to generate a second
non-captured cell-free DNA sample; detecting the second level of
aneuploidy in the second non-captured non-capture cell-free
DNA.
11. The method of claim 8, wherein a second level of at least one
genetic alteration in circulating tumor DNA (ctDNA) in the
biological sample isolated from the subject at the first time point
is not substantially different than a first level of the at least
one genetic alteration in circulating tumor DNA (ctDNA) in the
biological sample isolated from the subject at the first time
point.
12. The method of claim 1, wherein the biological sample obtained
from the subject at the first time point, the second time point, or
both comprises blood, plasma, serum, urine, cerebrospinal fluid,
saliva, sputum, broncho-alveolar lavage, bile, lymphatic fluid,
cyst fluid, stool, uterine lavage, vaginal fluids, ascites, and
combinations thereof.
13. The method of claim 1, wherein the targeted therapy is a kinase
inhibitor.
14-15. (canceled)
16. The method of claim 1, wherein the subject has been previously
administered a different treatment or targeted therapy and the
different treatment or targeted therapy was determined not to be
therapeutically effective.
17. (canceled)
18. The method of claim 17, further comprising administering a
therapeutic intervention to the subject.
19. (canceled)
20. The method of claim 1, wherein the cancer is selected from the
group consisting of: a head and neck cancer, a central nervous
system cancer, a lung cancer, a mesothelioma, an esophageal cancer,
a gastric cancer, a gall bladder cancer, a liver cancer, a
pancreatic cancer, a melanoma, an ovarian cancer, a small intestine
cancer, a colorectal cancer, a breast cancer, a sarcoma, a kidney
cancer, a bladder cancer, a uterine cancer, a cervical cancer, and
a prostate cancer.
21. The method of claim 20, wherein the cancer is a lung cancer,
and the lung cancer is non-small cell lung cancer.
22. The method of claim 20, wherein the cancer comprises a
population of cancer cells that harbor an EGFR mutation, a ERBB2
mutation, or both.
23. The method of claim 1, the second time point is between about 1
week to about 4 weeks after the first time point.
24-25. (canceled)
26. A method of determining response to a targeted therapy in a
subject having cancer, comprising: detecting a first level of at
least one genetic alteration in circulating tumor DNA (ctDNA) in a
biological sample isolated from the subject at a first time point;
detecting a second level of the at least one genetic alteration in
circulating tumor DNA (ctDNA) in a biological sample obtained from
the subject at a second time point, wherein the subject has
received at least one dose of the targeted therapy between the
first time point and the second time point; and identifying the
subject as responding to the targeted therapy when the second level
of the at least one genetic alteration is substantially increased
as compared to the first level of the at least one genetic
alteration.
27-28. (canceled)
29. A method of determining poor efficacy of a targeted therapy in
a subject having cancer, comprising: detecting a first cell-free
tumor load (cfTL) in a biological sample isolated from the subject
at a first time point; detecting a second cfTL in a biological
sample obtained from the subject at a second time point, wherein
the subject has received at least one dose of the targeted therapy
between the first time point and the second time point; and
identifying the targeted therapy as having poor efficacy in the
subject when the subject exhibits a second cfTL that is not
substantially reduced as compared to the first cfTL.
30-34. (canceled)
Description
TECHNICAL FIELD
[0002] The present disclosure relates generally to the field of
cancer. More specifically, this disclosure relates to non-invasive
in vitro methods for determining the efficacy of a targeted therapy
(e.g., a kinase inhibitor (KI)).
BACKGROUND
[0003] The management of oncogene-addicted cancer has been improved
by the development of targeted therapies that act against a variety
of cancer dependencies. However, therapeutic efficacy of targeted
therapies has been limited by incomplete pharmacological
suppression of tumors or through the selection of resistance
mutations in sub-clonal populations of tumor cells. Disease
monitoring using computed tomography (CT) imaging is the current
clinical practice for assessing response to targeted therapy, yet
this approach does not fully represent the molecular and pathologic
changes occurring in tumors during therapy.
SUMMARY
[0004] With the advent of precision oncology approaches, there is
an urgent need to develop improved methods for rapidly detecting
responses to targeted therapies. An ultrasensitive measure of
cell-free tumor burden was developed using targeted and whole
genome sequencing approaches to assess responses to tyrosine kinase
inhibitors in advanced lung cancer patients. Patients had a bimodal
distribution of cell-free circulating tumor DNA (ctDNA) one to
three weeks after therapy, with responders having nearly complete
elimination of ctDNA while non-responders had limited changes in
ctDNA. Changes in sequence alterations in ctDNA were detectable
within hours after treatment in responders. Patients with ctDNA
responses had improved progression-free survival (10.8 vs 2.0
months, P<0.001), which was detected on average 40 days earlier
and was as predictive as CT imaging. These analyses provide a rapid
approach for evaluating therapeutic outcomes to targeted therapies
and have important implications for the management of cancer
patients and development of new therapeutics.
[0005] In one aspect, provided herein are methods of predicting the
efficacy of a targeted therapy (e.g., a kinase inhibitor (e.g., a
tyrosine kinase inhibitor (TKI))) in a subject having been
previously diagnosed with cancer and having received at least one
dose of a targeted therapy (e.g., a kinase inhibitor (e.g., a
tyrosine kinase inhibitor (TKI))).
[0006] In some embodiments, provided herein are methods of
determining the efficacy of a targeted therapy in a subject having
cancer that include: detecting a first cell-free tumor load (cfTL)
in a biological sample isolated from the subject at a first time
point, detecting a second cfTL in a biological sample obtained from
the subject at a second time point, wherein the subject has
received at least one dose of the targeted therapy between the
first time point and the second time point, and identifying the
targeted therapy as being effective in the subject when the subject
exhibits a second cfTL that is reduced as compared to the first
cfTL.
[0007] In some embodiments of detecting a first and second cfTL
(e.g. a first and second cfTL at different time points), detecting
the first cfTL includes detecting a first level of the at least one
genetic alteration in circulating tumor DNA (ctDNA) in the
biological sample isolated from the subject at the first time
point, wherein the first cfTL corresponds to the first level of the
at least one genetic alteration, and detecting the second cfTL
includes detecting a second level of the at least one genetic
alteration in circulating tumor DNA (ctDNA) in the biological
sample isolated from the subject at the second time point, wherein
the second cfTL corresponds to the second level of the at least one
genetic alteration. In some embodiments, detecting the first level
of the at least one genetic alteration, detecting the second level
of the at least one genetic alteration, or both includes using a
method selected from the group consisting of: a targeted capture
method, a next-generation sequencing method, an array-based method,
and combinations thereof. In some embodiments, detecting the first
level of the at least one genetic alteration, detecting the second
level of the at least one genetic alteration, or both includes:
extracting cell-free DNA from blood; ligating a low complexity pool
of dual index barcode adapters to the cell-free DNA to generate a
plurality of barcode adapter-ligated cell-free DNA segments,
capturing the plurality of barcode adapter-ligated cell-free DNA
segments, sequencing the plurality of captured barcode
adapter-ligated cell-free DNA segments, aligning the sequenced
plurality of captured barcode adapter-ligated cell-free DNA
segments to a reference genome, and identifying sequence
alterations using aligned sequences of multiple distinct molecules
containing identical redundant changes.
[0008] In some embodiments of any of the methods provided herein
that includes detecting at least one genetic alteration, the at
least one genetic is a mutation is in an EGFR gene, an ERBB2 gene,
or both. In some embodiments of any of the methods provided herein
that includes detecting at least one genetic alteration, the at
least one genetic alteration is a T790M mutation in the EGFR
gene.
[0009] In some embodiments of any of the methods provided herein
that includes detecting a first and second level of at least one
genetic alteration, the second level of the at least one genetic
alteration of ctDNA is at least about 90% lower than the first
level of the at least one genetic alteration of ctDNA.
[0010] In some embodiments of detecting a first and second cfTL
(e.g. a first and second cfTL at different time points), detecting
the first cfTL comprises detecting a first level of aneuploidy in
the biological sample isolated from the subject at the first time
point, wherein the first cfTL corresponds to the first level of
aneuploidy, and detecting the second cfTL comprises detecting a
second level of aneuploidy in the biological sample isolated from
the subject at the second time point, wherein the second cfTL
corresponds to the second level of aneuploidy. In some embodiments,
detecting the first level of aneuploidy, detecting the second level
of aneuploidy, or both includes: performing digital karyotyping,
next generation sequencing, array-based methods, and combinations
thereof. In some embodiments, the method further includes: a)
extracting a first sample of cell-free DNA from blood at the first
time point, ligating a low complexity pool of dual index barcode
adapters to the first cell-free DNA sample to generate a first
plurality of barcode adapter-ligated cell-free DNA segments,
capturing the first plurality of barcode adapter-ligated cell-free
DNA segments, eluting the non-captured cell-free DNA to generate a
first non-captured cell-free DNA sample, detecting the first level
of aneuploidy in the first non-captured non-capture cell-free DNA;
and b) extracting a second sample of cell-free DNA from blood at
the second time point, ligating a low complexity pool of dual index
barcode adapters to the cell-free DNA to generate a plurality of
barcode adapter-ligated cell-free DNA segments, capturing the
plurality of barcode adapter-ligated cell-free DNA segments,
eluting the non-captured cell-free DNA to generate a second
non-captured cell-free DNA sample, detecting the second level of
aneuploidy in the second non-captured non-capture cell-free
DNA.
[0011] In some embodiments of any of the methods provided herein in
which a first and second level of circulating tumor DNA (ctDNA) is
detected, a second level of at least one genetic alteration in
circulating tumor DNA (ctDNA) in the biological sample isolated
from the subject at the first time point is not substantially
different than a first level of the at least one genetic alteration
in circulating tumor DNA (ctDNA) in the biological sample isolated
from the subject at the first time point.
[0012] In some embodiments of any of the methods provided herein, a
biological sample obtained from the subject at the first time
point, the second time point, or both comprises blood, plasma,
serum, urine, cerebrospinal fluid, saliva, sputum, broncho-alveolar
lavage, bile, lymphatic fluid, cyst fluid, stool, uterine lavage,
vaginal fluids, ascites, and combinations thereof.
[0013] In some embodiments of any of the methods provided herein,
the targeted therapy is a kinase inhibitor. In some embodiments,
the kinase inhibitor is a tyrosine kinase inhibitor. In some
embodiments, the kinase inhibitor is selected from the group
consisting of: afatinib, crizotinib, erlotinib, gefitinib,
osimertinib, and combinations thereof.
[0014] In some embodiments of any of the methods provided herein,
the subject has been previously administered a different treatment
or targeted therapy and the different treatment or targeted therapy
was determined not to be therapeutically effective. In some
embodiments of any of the methods provided herein, the method
further includes administering one or more additional doses of the
targeted therapy identified as being effective to the subject.
[0015] In some embodiments of any of the methods provided herein,
the method further includes administering a therapeutic
intervention to the subject. In some embodiments, the therapeutic
intervention is selected from the group consisting of: a different
targeted therapy, an antibody, an adoptive T cell therapy, a
chimeric antigen receptor (CAR) T cell therapy, an antibody-drug
conjugate, a cytokine therapy, a cancer vaccine, a checkpoint
inhibitor, radiation therapy, surgery, a chemotherapeutic agent,
and combinations thereof.
[0016] In some embodiments of any of the methods provided herein, a
subject has a cancer selected from the group consisting of: a head
and neck cancer, a central nervous system cancer, a lung cancer, a
mesothelioma, an esophageal cancer, a gastric cancer, a gall
bladder cancer, a liver cancer, a pancreatic cancer, a melanoma, an
ovarian cancer, a small intestine cancer, a colorectal cancer, a
breast cancer, a sarcoma, a kidney cancer, a bladder cancer, a
uterine cancer, a cervical cancer, and a prostate cancer. In some
embodiments, the cancer is a lung cancer, and the lung cancer is
non-small cell lung cancer. In some embodiments, the cancer
comprises a population of cancer cells that harbor an EGFR
mutation, a ERBB2 mutation, or both.
[0017] In some embodiments of any of the methods provided herein in
which cfTL is determined a first and second time point, the second
time point is between about 1 week to about 4 weeks after the first
time point. In some embodiments, the second time point is about 16
days after the first time point. In some embodiments, the second
time point is about 6 days after the first time point.
[0018] In some embodiments, provided herein are methods of
determining response to a targeted therapy in a subject having
cancer that include: detecting a first level of at least one
genetic alteration in circulating tumor DNA (ctDNA) in a biological
sample isolated from the subject at a first time point, detecting a
second level of the at least one genetic alteration in circulating
tumor DNA (ctDNA) in a biological sample obtained from the subject
at a second time point, wherein the subject has received at least
one dose of the targeted therapy between the first time point and
the second time point, and identifying the subject as responding to
the targeted therapy when the second level of the at least one
genetic alteration is substantially increased as compared to the
first level of the at least one genetic alteration. In some
embodiments, the second time point is about 4 to about 12 hours
after the first time point. In some embodiments, the second time
point is about 4 to about 12 hours after the first time point.
[0019] In some embodiments, provided herein are methods of
determining poor efficacy of a targeted therapy in a subject having
cancer that include: detecting a first cell-free tumor load (cfTL)
in a biological sample isolated from the subject at a first time
point, detecting a second cfTL in a biological sample obtained from
the subject at a second time point, wherein the subject has
received at least one dose of the targeted therapy between the
first time point and the second time point, and identifying the
targeted therapy as having poor efficacy in the subject when the
subject exhibits a second cfTL that is not substantially reduced as
compared to the first cfTL. In some embodiments, the second time
point is between about 1 week to about 4 weeks after the first time
point. In some embodiments, the subject is identified as having
poor prognosis when the targeted therapy was identified as having
poor efficacy. In some embodiments, the poor prognosis is selected
from the group consisting of: shorter progression-free survival,
lower overall survival, and combinations thereof.
[0020] In some embodiments of any of the methods of determining
poor efficacy of a targeted therapy in a subject having cancer
provided herein, the method further includes administering a
therapeutic intervention to the subject, wherein the therapeutic
intervention is not the targeted therapy. In some embodiments, the
therapeutic intervention is selected from: a different targeted
therapy, an antibody, an adoptive T cell therapy, a chimeric
antigen receptor (CAR) T cell therapy, an antibody-drug conjugate,
a cytokine therapy, a cancer vaccine, a checkpoint inhibitor,
radiation therapy, surgery, a chemotherapeutic agent, and
combinations thereof.
[0021] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control.
[0022] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
BRIEF DESCRIPTION OF DRAWINGS
[0023] FIG. 1 is a schematic overview of cfTL determination and
prediction of therapeutic response. Liquid biopsies from metastatic
non-small-cell lung cancer (mNSCLC) patients undergoing treatment
with tyrosine kinase inhibition (TKI) were analyzed at baseline and
6-22 days after treatment. The TEC-Seq approach was used to
directly identify sequence alterations across 58 genes encompassing
80,930 bases sequenced to >30,000.times. coverage, and
whole-genome approaches were used to identify copy number changes
in cfDNA. Cell-free tumor load (cfTL) was determined as the mutant
allele fraction of the most abundant alteration in a clone targeted
by TKI for patients with detected sequence alterations, or as the
presence or absence of aneuploidy based on PA score in patients
without detectable sequence alterations. Prediction of therapeutic
response to targeted therapy based on ctDNA dynamics was assessed
through changes in cfTL from baseline to day 6-22 after treatment
whereas response assessment through CT imaging was performed 4-7
weeks after treatment.
[0024] FIG. 2A is line graphs showing ctDNA changes for a responder
patient (CGPLLU12) treated with osimertinib (left) and a
non-responder patient (CGPLLU244) treated with (right). Mutant
allele fractions of clones identified in cfDNA through the TEC-Seq
approach are shown for each time point analyzed with the ctDNA
clone representing cfTL shown in bright green and treatment
initiation highlighted with a red arrow. RECIST 1.1 sum of longest
diameters (SLD, gray boxes) were measured from CT scans at
intervals during therapy.
[0025] FIG. 2B is line graphs showing copy number changes
identified in cfDNA from analyses of whole-genome data at each time
point analyzed as Z scores (burgundy dots) for each chromosome arm
and PA scores (orange diamonds) for a responder patient (CGPLLU2)
treated with osimertinib (left) and a non-responder patient
(CGPLLU244) treated with (right).
[0026] FIG. 2C shows computer tomography (CT) images showing
representative tumor lesions (circled in red) at different time
points for a responder patient (CGPLLU12) treated with osimertinib
(left) and a non-responder patient (CGPLLU244) treated with (right)
FIG. 3A is a line graph showing changes in cfTL (P=0.002:
responders, P=0.625: non-responders, Wilcoxon signed rank test) in
radiographic responders (blue) and radiographic non-responders
(orange) from baseline to day 6-22 post treatment.
[0027] FIG. 3B is a line graph showing PA score (P=0.002:
responders, P=0.875: non-responders, Wilcoxon signed rank test) in
radiographic responders (blue) and radiographic non-responders
(orange) from baseline to day 6-22 post treatment.
[0028] FIG. 3C is line graph showing the number of mutations
(P=0.006: responders, P=1.000: non-responders, Wilcoxon signed rank
test) in radiographic responders (blue) and radiographic
non-responders (orange) from baseline to day 6-22 post
treatment.
[0029] FIG. 4A is a line graph showing changes in the levels of
ctDNA for six patients at baseline and at four to twelve hours
after the initiation of targeted therapy. Emerging ctDNA
alterations are depicted in red.
[0030] FIG. 4B is a line graph showing changes in the levels of
cfDNA extracted from six patients at baseline and at four to twelve
hours after the initiation of targeted therapy. Emerging ctDNA
alterations are depicted in red.
[0031] FIG. 5A is a bar graph showing changes in cfTL from baseline
to days 6-22 post treatment clustering patients with reduction of
cfTL.gtoreq.98% as ctDNA responders and .ltoreq.98% as ctDNA
non-responders.
[0032] FIG. 5B is a graph showing cfTL at days 6-22 post treatment
and PFS for patients analyzed with radiographic assessment in the
right column denoting partial response (PR), stable disease (SD),
unmeasureable disease (*), or progressive disease (PD). cfTL levels
at day 6-22 were significantly different between ctDNA responders
and non-responders (P<0.01, Wilcoxon signed rank test).
[0033] FIG. 5C is a Kaplan-Meier curve showing PFS for ctDNA
responders and non-responders (P<0.001, Mantel-Cox log rank
test).
[0034] FIG. 5D is a line graph showing time to response assessment
based on CT scan (orange) and analyses of ctDNA (blue) with mean
times to assessment shown in dotted lines (P<0.0001, Wilcoxon
signed rank test).
[0035] FIG. 6A shows line graphs showing mutant allele fractions of
clones identified in cfDNA through the TEC-Seq approach for each
time point analyzed with treatment initiation highlighted with a
red arrow (top), and copy number changes identified in cfDNA from
analyses of whole-genome data at each time point analyzed as Z
scores (burgundy dots) for each chromosome arm and PA scores
(orange diamonds) (bottom) for patient (CGPLLU88). RECIST 1.1 sum
of longest diameters (SLD, gray boxes) were measured from CT scans
at intervals during therapy (top).
[0036] FIG. 6B shows line graphs showing mutant allele fractions of
clones identified in cfDNA through the TEC-Seq approach for each
time point analyzed with treatment initiation highlighted with a
red arrow (top), and copy number changes identified in cfDNA from
analyses of whole-genome data at each time point analyzed as Z
scores (burgundy dots) for each chromosome arm and PA scores
(orange diamonds) (bottom) for patient (CGPLLU99). RECIST 1.1 sum
of longest diameters (SLD, gray boxes) were measured from CT scans
at intervals during therapy (top).
[0037] FIG. 6C shows line graphs showing mutant allele fractions of
clones identified in cfDNA through the TEC-Seq approach for each
time point analyzed with treatment initiation highlighted with a
red arrow (top), and copy number changes identified in cfDNA from
analyses of whole-genome data at each time point analyzed as Z
scores (burgundy dots) for each chromosome arm and PA scores
(orange diamonds) (bottom) for patient (CGPLLU315). RECIST 1.1 sum
of longest diameters (SLD, gray boxes) were measured from CT scans
at intervals during therapy (top).
[0038] FIG. 6D shows line graphs showing mutant allele fractions of
clones identified in cfDNA through the TEC-Seq approach for each
time point analyzed with treatment initiation highlighted with a
red arrow (top), and copy number changes identified in cfDNA from
analyses of whole-genome data at each time point analyzed as Z
scores (burgundy dots) for each chromosome arm and PA scores
(orange diamonds) (bottom) for patient (CGPLLU86). RECIST 1.1 sum
of longest diameters (SLD, gray boxes) were measured from CT scans
at intervals during therapy (top).
[0039] FIG. 6E shows line graphs showing mutant allele fractions of
clones identified in cfDNA through the TEC-Seq approach for each
time point analyzed with treatment initiation highlighted with a
red arrow (top), and copy number changes identified in cfDNA from
analyses of whole-genome data at each time point analyzed as Z
scores (burgundy dots) for each chromosome arm and PA scores
(orange diamonds) (bottom) for patient (CGPLLU89). RECIST 1.1 sum
of longest diameters (SLD, gray boxes) were measured from CT scans
at intervals during therapy (top).
[0040] FIG. 6F shows line graphs showing mutant allele fractions of
clones identified in cfDNA through the TEC-Seq approach for each
time point analyzed with treatment initiation highlighted with a
red arrow (top), and copy number changes identified in cfDNA from
analyses of whole-genome data at each time point analyzed as Z
scores (burgundy dots) for each chromosome arm and PA scores
(orange diamonds) (bottom) for patient (CGPLLU319). RECIST 1.1 sum
of longest diameters (SLD, gray boxes) were measured from CT scans
at intervals during therapy (top).
[0041] FIG. 6G shows line graphs showing mutant allele fractions of
clones identified in cfDNA through the TEC-Seq approach for each
time point analyzed with treatment initiation highlighted with a
red arrow (top), and copy number changes identified in cfDNA from
analyses of whole-genome data at each time point analyzed as Z
scores (burgundy dots) for each chromosome arm and PA scores
(orange diamonds) (bottom) for patient (CGPLLU14). RECIST 1.1 sum
of longest diameters (SLD, gray boxes) were measured from CT scans
at intervals during therapy (top).
[0042] FIG. 6H shows line graphs showing mutant allele fractions of
clones identified in cfDNA through the TEC-Seq approach for each
time point analyzed with treatment initiation highlighted with a
red arrow (top), and copy number changes identified in cfDNA from
analyses of whole-genome data at each time point analyzed as Z
scores (burgundy dots) for each chromosome arm and PA scores
(orange diamonds) (bottom) for patient (CGPLLU324). RECIST 1.1 sum
of longest diameters (SLD, gray boxes) were measured from CT scans
at intervals during therapy (top).
[0043] FIG. 6I shows line graphs showing mutant allele fractions of
clones identified in cfDNA through the TEC-Seq approach for each
time point analyzed with treatment initiation highlighted with a
red arrow (top), and copy number changes identified in cfDNA from
analyses of whole-genome data at each time point analyzed as Z
scores (burgundy dots) for each chromosome arm and PA scores
(orange diamonds) (bottom) for patient (CGPLLU97). RECIST 1.1 sum
of longest diameters (SLD, gray boxes) were measured from CT scans
at intervals during therapy (top).
[0044] FIG. 6J shows line graphs showing mutant allele fractions of
clones identified in cfDNA through the TEC-Seq approach for each
time point analyzed with treatment initiation highlighted with a
red arrow (top), and copy number changes identified in cfDNA from
analyses of whole-genome data at each time point analyzed as Z
scores (burgundy dots) for each chromosome arm and PA scores
(orange diamonds) (bottom) for patient (CGPLLU245). RECIST 1.1 sum
of longest diameters (SLD, gray boxes) were measured from CT scans
at intervals during therapy (top).
[0045] FIG. 6K shows line graphs showing mutant allele fractions of
clones identified in cfDNA through the TEC-Seq approach for each
time point analyzed with treatment initiation highlighted with a
red arrow (top), and copy number changes identified in cfDNA from
analyses of whole-genome data at each time point analyzed as Z
scores (burgundy dots) for each chromosome arm and PA scores
(orange diamonds) (bottom) for patient (CGPLLU246). RECIST 1.1 sum
of longest diameters (SLD, gray boxes) were measured from CT scans
at intervals during therapy (top).
[0046] FIG. 6L shows line graphs showing mutant allele fractions of
clones identified in cfDNA through the TEC-Seq approach for each
time point analyzed with treatment initiation highlighted with a
red arrow (top), and copy number changes identified in cfDNA from
analyses of whole-genome data at each time point analyzed as Z
scores (burgundy dots) for each chromosome arm and PA scores
(orange diamonds) (bottom) for patient (CGPLLU294). RECIST 1.1 sum
of longest diameters (SLD, gray boxes) were measured from CT scans
at intervals during therapy (top).
[0047] FIG. 6M shows line graphs showing mutant allele fractions of
clones identified in cfDNA through the TEC-Seq approach for each
time point analyzed with treatment initiation highlighted with a
red arrow (top), and copy number changes identified in cfDNA from
analyses of whole-genome data at each time point analyzed as Z
scores (burgundy dots) for each chromosome arm and PA scores
(orange diamonds) (bottom) for patient (CGPLLU18). RECIST 1.1 sum
of longest diameters (SLD, gray boxes) were measured from CT scans
at intervals during therapy (top).
[0048] FIG. 7 is a graph showing concordance between alterations
observed with TEC-seq in the plasma and clinical NGS analyses in
the tumor tissue or plasma. The presence of each alteration in
matched tumor tissue or plasma specimen evaluated with clinical NGS
tests are indicated with dark blue and light blue dots respectively
whereas nonconcordant mutations are indicated in orange.
[0049] FIG. 8 is a plot showing the timeline of ctDNA analyses, CT
assessments and treatment response. Interval between cfTL response
assessment at day 6-22 (colored circles) and CT scan response
(green squares) depicts the lead time between ctDNA and imaging
analyses. Interval between treatment start and CT scan progression
(red squares) depicts progression-free survival.
[0050] FIG. 9 is a graph showing the change in RECIST 1.1 SLD at
median times of radiographic assessment for ctDNA responders (blue
lines) and non-responders (orange lines) with the window of cfTL
assessment shown in the dotted bracket.
[0051] FIG. 10 is a plot showing the correlation of cfTL at day
6-22 and percent reduction in RECIST SLD on initial CT scan. Inset
bar chart depicts cfTL at day 6-22 for ctDNA responders (blue) and
ctDNA non-responders (orange).
[0052] FIG. 11 is a Kaplan-Meier curve showing PFS for patients
(n=14) with partial response (blue line) or progressive disease
(orange line) based on radiographic imaging obtained 5-7 weeks
after treatment initiation.
DETAILED DESCRIPTION
[0053] As used herein, the word "a" or "an" before a noun
represents one or more of the particular noun. For example, the
phrase "an immunotherapy" encompasses "one or more
immunotherapies."
[0054] As used herein, the term "about" means approximately, in the
region of, roughly, or around. When used in conjunction with a
numerical range, the term "about" modifies that range by extending
the boundaries above and below the numerical values set forth. In
general, the term "about" is used herein to modify a numerical
value above and below the stated value by a variance of 10%.
[0055] As used herein, the term "subject" means a vertebrate,
including any member of the class mammalia, including humans,
domestic and farm animals, and zoo, sports or pet animals, such as
mouse, rabbit, pig, sheep, goat, cattle, horse (e.g., race horse),
and higher primates. In some embodiments, the subject is a human.
In some embodiments, the subject is a human harboring a cancer
cell. In some embodiments, the subject is a human harboring a
cancer cell, but who is not known to harbor the cancer cell.
[0056] The term "treat(ment)" is used herein to denote delaying the
onset of, inhibiting, alleviating the effects of, or prolonging the
life of a patient suffering from, a condition, e.g., cancer.
[0057] The terms "effective amount" and "amount effective to treat"
as used herein, refer to an amount or concentration of a
composition or treatment described herein, e.g., a targeted therapy
(e.g., any targeted therapy described herein), utilized for a
period of time (including acute or chronic administration and
periodic or continuous administration) that is effective within the
context of its administration for causing an intended effect or
physiological outcome. For example, effective amounts of a targeted
therapy (e.g., any targeted therapy described herein) for use in
the present disclosure include, for example, amounts that inhibit
the growth of cancer, e.g., tumors and/or tumor cells, improve,
delay tumor growth, improve survival for a patient suffering from
or at risk for cancer, and improve the outcome of other cancer
treatments. As another example, effective amounts of a targeted
therapy (e.g., any of the targeted therapies described herein) can
include amounts that advantageously affect a tumor
microenvironment, e.g., the cell-free tumor load (cfTL), the level
of at least one genetic alteration of circulating tumor DNA (ctDNA)
(e.g., a mutation), and/or the level of aneuploidy in the
ctDNA.
[0058] The terms "a reduced level" or a "decreased level" refer to
a reduction or decrease in the level of a particular substance or
particular substances (e.g., cfTL and/or ctDNA) of at least about
2-fold (e.g., at least about 4-fold, at least about 6-fold, at
least about 8-fold, at least about 10-fold, at least about 12-fold,
at least about 14-fold, at least about 20-fold) as compared to a
reference level or value.
[0059] In some embodiments, a reduced level is a reduction of or
decrease in a second level of a particular substance(s) or a
particular parameter(s) (e.g., cfTL and/or ctDNA) of at least about
1% (e.g., at least about 2%, at least about 4%, at least about 6%,
at least about 8%, at least about 10%, at least about 12%, at least
about 14%, at least about 16%, at least about 18%, at least about
20%, at least about 22%, at least about 24%, at least about 26%, at
least about 28%, at least about 30%, at least about 40%, at least
about 45%, at least about 50%, at least about 60%, at least about
65%, at least about 70%, at least about 75%, at least about 80%, at
least about 85%, at least about 90%, at least about 95%, or at
least about 99%) as compared to the first level of the particular
substance or particular parameter.
[0060] The terms "an increased level" or a "higher level" refer to
an increase of at least about 2-fold (e.g., at least about 4-fold,
at least about 6-fold, at least about 8-fold, at least about
10-fold, at least about 12-fold, at least about 14-fold, at least
about 20-fold, or more) of a particular substance(s) or a
particular parameter(s) (e.g., cfTL and/or ctDNA). In some
embodiments, an increased level of at least one genetic alteration
present in ctDNA (e.g., at least two, at least three, at least
four, at least five, at least six, at least seven, at least eight,
at least nine, at least ten, at least twelve, at least fifteen, at
least twenty, or more) is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, or 30-fold higher as compared to a first or reference level of
the genetic alteration present in ctDNA.
[0061] In some embodiments, an increased level of at least one
genetic alteration present in ctDNA is an increase of at least
about 1% (e.g., at least about 2%, at least about 4%, at least
about 6%, at least about 8%, at least about 10%, at least about
12%, at least about 14%, at least about 16%, at least about 18%, at
least about 20%, at least about 22%, at least about 24%, at least
about 26%, at least about 28%, at least about 30%, at least about
40%, at least about 45%, at least about 50%, at least about 60%, at
least about 65%, at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
95%, or at least about 99%) of a second level of the at least one
genetic alteration as compared to a first or reference level of the
at least one genetic alteration.
[0062] The terms "not substantially reduced" or "not substantially
decreased" refer to clinically insignificant changes (e.g., a
reduction or decrease) in the second level of a particular
substance(s) or a particular parameter(s) (e.g., cfTL) as compared
to the first level of the particular substance or particular
parameter. In contrast, the terms "substantially reduced" or
"substantially decreased" refer to clinically significant changes
in the second level of a particular substance(s) or a particular
parameter(s) (e.g., cfTL) as compared to the first level of the
particular substance or particular parameter.
[0063] In some embodiments, a not substantially reduced second
level of a detected particular substance(s) or a particular
parameter(s) (e.g., cfTL) is a reduction or decrease of less than
about 10% (e.g., less than about 9%, less than about 8%, less than
about 7%, less than about 6%, less than about 5%, less than about
4%, less than about 3%, less than about 2%, less than about 1%,
less than about 0.5%, less than about 0.25%, less than about 0.2%,
less than about 0.1%, less than about 0.05%, less than about 0.01%)
as compared to the first detected level of the particular substance
or particular parameter.
[0064] In some embodiments, a not substantially reduced level of a
particular detected substance(s) or a particular detected
parameter(s) (e.g., cfTL) is an increase of at least about 0.5%
(e.g., at least about 1%, at least about 2%, at least about 4%, at
least about 6%, at least about 8%, at least about 10%, at least
about 12%, at least about 14%, at least about 16%, at least about
18%, at least about 20%, at least about 22%, at least about 24%, at
least about 26%, at least about 28%, at least about 30%, at least
about 40%, at least about 45%, at least about 50%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 90%, at least
about 95%, or at least about 99%) in the second detected level of
the substance or parameter as compared to the first detected level
of the substance or parameter.
[0065] The terms "substantially increased" refers to clinically
significant changes (e.g., an increase) in the second level of a
particular substance or particular substances (e.g., at least one
genetic alteration of ctDNA) as compared to the first level of the
particular substance or particular substances. In contrast, the
term "not substantially increased" refers to clinically
insignificant changes in the second level of a particular
substance(s) or a particular parameter(s) (e.g., cfTL) as compared
to the first level of the particular substance or particular
parameter.
[0066] In some embodiments, a not substantially increased second
level of a particular substance(s) or particular parameter(s)
(e.g., at least one genetic alteration of ctDNA) is an increase in
levels of at least about 0.5% (e.g., at least about 1%, at least
about 2%, at least about 4%, at least about 6%, at least about 8%,
at least about 10%, at least about 12%, at least about 14%, at
least about 16%, at least about 18%, at least about 20%, at least
about 22%, at least about 24%, at least about 26%, at least about
28%, at least about 30%, at least about 40%, at least about 45%, at
least about 50%, at least about 60%, at least about 65%, at least
about 70%, at least about 75%, at least about 80%, at least about
85%, at least about 90%, at least about 95%, or at least about 99%)
as compared to the first level of particular substance or
particular parameter.
[0067] A "chemotherapeutic agent" refers to a chemical compound
useful in the treatment of a cancer. Chemotherapeutic agents
include, e.g., "anti-hormonal agents" or "endocrine therapeutics"
which act to regulate, reduce, block or inhibit the effects of
hormones that can promote the growth of cancer. Additional classes,
subclasses and examples of chemotherapeutic agents are known in the
art.
[0068] The terms "acquired resistance" and "resistance" when used
in reference to a targeted therapy refer to a subsequent state of
decreased effectiveness of the targeted therapy (e.g., when the
targeted therapy was initially effective). As will be appreciated
by those of ordinary skill in the art, resistance to targeted
therapy can arise in a subject receiving targeted therapy treatment
when a tumor cell in the subject develops a mutation or other
molecular lesion that render the tumor cell resistant to the
targeted therapy. In some embodiments, when a subject develops
resistance to a first targeted therapy, a therapeutic intervention
can be administered to the subject (e.g., the therapeutic
intervention can be different from the first targeted therapy,
including but not limited to, a different targeted therapy, an
immunotherapy, a chemotherapy, a surgery, or any of the variety of
other therapeutic interventions disclosed herein).
[0069] Skilled practitioners will appreciate that a subject can be
diagnosed, e.g., by a medical professional, e.g., a physician or
nurse (or veterinarian, as appropriate for the patient being
diagnosed), as suffering from or at risk for a condition described
herein, e.g., cancer, using any method known in the art, e.g., by
assessing a subject's medical history, performing diagnostic tests,
and/or by employing imaging techniques.
[0070] Skilled practitioners will also appreciate that treatment
need not be administered to a subject by the same individual who
diagnosed the subject (or the same individual who prescribed the
treatment for the subject). Treatment can be administered (and/or
administration can be supervised), e.g., by the diagnosing and/or
prescribing individual, and/or any other individual, including the
subject her/himself (e.g., where the patient is capable of
self-administration).
[0071] The management of oncogene-addicted cancer has been improved
by the development of targeted therapies that act against a variety
of cancer dependencies (1, 2). However, therapeutic efficacy of
targeted therapies has been limited by incomplete pharmacological
suppression of tumors or through the selection of resistance
mutations in sub-clonal populations of tumor cells. Disease
monitoring using computed tomography (CT) imaging is the current
clinical practice for assessing response to targeted therapy, yet
this approach does not fully represent the molecular and pathologic
changes occurring in tumors during therapy. Repeat tissue biopsies
of accessible cancer lesions have been used to provide insights
into therapeutic decision-making but rarely capture the complexity
of intra- and inter-tumoral heterogeneity and are invasive
procedures with potential complications. Theoretically, the ability
to non-invasively track specific clonal populations of tumor cells
through time has the potential to rapidly and dynamically inform
therapy sequence and combinatorial strategies. However, there are
currently no approved or clinically recognized non-invasive
molecularly defined strategies to assess early drug responsiveness
or adaptive resistance in cancer patients before radiographic
progression.
[0072] Cell-free circulating tumor DNA (ctDNA) is released from
tumor cells into the circulation and has been detected in patients
with early and late stage cancers (3-8). A key challenge of liquid
biopsy approaches has been developing methods to detect and
characterize small fractions of ctDNA in large populations of total
cell-free DNA. A variety of studies have focused on changes in
ctDNA during the course of therapy, but have largely focused on the
analysis of specific or limited number of alterations that may only
represent specific sub-clones of the tumor (9-18). More recent
studies have used panels of commonly mutated driver genes to allow
detection of multiple driver clones, typically at the time of
diagnosis (4, 6, 19-21). However, no study has yet assessed the
clinical value of a comprehensive genome-wide analysis of ctDNA
alterations to evaluate tumor burden at very early time points
following commencement of targeted therapy.
[0073] In some embodiments, provided herein are ultrasensitive
liquid biopsy approaches that can be used to evaluate patients with
advanced non-small cell lung cancer (NSCLC) who have tumor
responses or progression on kinase inhibitors (e.g., tyrosine
kinase inhibitors). Non-limiting examples of tyrosine kinase
inhibitors include afatinib, a second-generation inhibitor of the
epidermal growth factor receptor (EGFR) and erb-b2 receptor
tyrosine kinase 2 (ERBB2) (22, 23) and osimertinib, a
third-generation tyrosine kinase inhibitor targeting EGFR with
activating and resistance (T790M) mutations (24, 25). In some
embodiments, methods provided herein can be used to assay rapid
changes and the overall levels in the amounts of ctDNA that can
serve as real-time and predictive biomarkers of patient outcome to
a targeted cancer therapy.
[0074] A dramatic reduction of cell-free tumor load was seen in
patients that were clinical responders, at time points that were
within 6-22 days after initiation of therapy. Remarkably, one
patient was classified with non-measurable disease at baseline and
another with stable disease at first imaging evaluation experienced
a complete clearance of ctDNA a few days after TKI initiation.
These examples reflect the utility of ctDNA for addressing current
unmet clinical needs for real time biomarkers of response and
evolution of tumor burden. The tiered complementary approach has
the benefit of incorporating sequence mutations in cfDNA that have
both qualitative and quantitative characteristics in the type and
level of detected alterations, while chromosomal changes add
quantitative assessment of genome-wide alterations that are
typically present in cancer.
[0075] Analysis of extremely early time points within hours after
initiation of therapy identified the emergence of new tumor-derived
mutations in most responding patients. The detection of new
mutations and increase in the levels of existing alterations at
this time provided insight into rapid release of ctDNA observed
during apoptotic cell death. These analyses open the possibility of
extremely early detection of response to targeted therapies.
[0076] In some embodiments, provided herein is a new paradigm in
cancer therapeutics and drug development in which cfTL molecular
response criteria may be used to provide insight into clinical
endpoints including overall survival and progression-free survival.
Given the heterogeneity of metastatic disease, various cfTL
approaches described herein have the advantage of measuring overall
tumor burden of clonal populations during selective pressure of
targeted therapies. For patients without molecular response, liquid
or tissue biopsies can provide additional information related to
mechanisms of resistance and provide a context to consider other
therapeutic strategies. In some embodiments, cfTL monitoring
provides an early biomarker for studies of novel targeted therapies
both for established and new molecular targets. Without wishing to
be bound by theory, it is thought that combining cfTL response
information with early pharmacokinetic data will ultimately provide
the biologically effective dose needed for an individual's cancer
rather than a maximally tolerated dose.
Methods of Determining Efficacy of a Targeted Therapy
[0077] Provided herein are methods of determining the efficacy of a
targeted therapy (e.g., any targeted therapy described herein
(e.g., a kinase inhibitor)) in a subject that include: detecting a
first cell-free tumor load (cfTL) in a biological sample isolated
from the subject at a first time point; detecting a second cfTL in
a biological sample obtained from the subject at a second time
point, wherein the subject has received at least one dose of the
targeted therapy between the first time point and the second time
point; and identifying the targeted therapy as being effective in
the subject when the subject exhibits a second cfTL that is reduced
as compared to the first cfTL.
[0078] In some embodiments of any of the methods described herein,
detecting the first cfTL includes detecting a first level of at
least one genetic alteration present in circulating tumor DNA
(ctDNA) in the biological sample isolated from the subject at the
first time point, wherein the first cfTL corresponds to the first
level of the at least one genetic alteration; and detecting the
second cfTL comprises detecting a second level of the at least one
genetic alteration present in circulating tumor DNA (ctDNA) in the
biological sample isolated from the subject at the second time
point, wherein the second cfTL corresponds to the second level of
the at least one genetic alteration.
[0079] In some embodiments of any of the methods described herein,
detecting the first cfTL comprises detecting a first level of
aneuploidy in the biological sample isolated from the subject at
the first time point, wherein the first cfTL corresponds to the
first level of aneuploidy; and detecting the second cfTL comprises
detecting a second level of aneuploidy in the biological sample
isolated from the subject at the second time point, wherein the
second cfTL corresponds to the second level of aneuploidy.
[0080] In some embodiments, a targeted therapy is determined to be
effective when the level of at least one genetic alteration present
in circulating tumor DNA (ctDNA) identified at the second time
point is decreased by at least about 2-fold, at least about 3-fold,
at least about 4-fold, at least about 5-fold, at least about
6-fold, at least about 7-fold, at least about 8-fold, at least
about 9-fold, or at least about 10-fold or more compared to the
level of the at least one genetic alteration of circulating tumor
DNA (ctDNA) identified at the first time point.
[0081] In some embodiments, a targeted therapy is determined to be
effective when the level of at least one genetic alteration present
in circulating tumor DNA (ctDNA) identified at the second time
point is at least about 25% (e.g., at least about 30%, at least
about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 91%, at least about 92%, at least about
93%, at least about 94%, at least about 95%, at least about 96%, at
least about 97%, at least about 98%, or at least about 99%) lower
than the level of the at least one genetic alteration of ctDNA
identified at the first time point.
[0082] In some embodiments, a targeted therapy is determined to be
effective when the circulating tumor DNA (ctDNA) is not observed at
the second time point.
[0083] Alternatively, a targeted therapy is determined to be
effective when the level of aneuploidy identified at the second
time point is at least about 25% (e.g., at least about 30%, at
least about 40%, at least about 45%, at least about 50%, at least
about 55%, at least about 60%, at least about 65%, at least about
70%, at least about 75%, at least about 80%, at least about 85%, at
least about 90%, at least about 91%, at least about 92%, at least
about 93%, at least about 94%, at least about 95%, at least about
96%, at least about 97%, at least about 98%, or at least about 99%)
lower than the first of aneuploidy identified at the first time
point.
[0084] In some embodiments, a targeted therapy is determined not to
be effective (e.g., the targeted therapy has poor efficacy) when
the amount of at least one genetic alteration present in
circulating tumor DNA (ctDNA) identified at the second time point
is not substantially decreased (e.g., is less than about 10%, less
than about 9%, less than about 8%, less than about 7%, less than
about 6%, less than about 5%, less than about 4%, less than about
3%, less than about 2%, less than about 1%, less than about 0.5%,
less than about 0.25%, less than about 0.2%, less than about 0.1%,
less than about 0.05%, or less than about 0.01%) as compared to the
amount of the at least one genetic alteration present in
circulating tumor DNA (ctDNA) identified at the first time
point.
[0085] In some embodiments, a targeted therapy is determined not to
be effective (e.g., the targeted therapy has poor efficacy) when
the level of aneuploidy identified at the second time point is not
substantially decreased (e.g., is less than about 10%, less than
about 9%, less than about 8%, less than about 7%, less than about
6%, less than about 5%, less than about 4%, less than about 3%,
less than about 2%, less than about 1%, less than about 0.5%, less
than about 0.25%, less than about 0.2%, less than about 0.1%, less
than about 0.05%, or less than about 0.01%) as compared to the
level of aneuploidy identified at the first time point.
[0086] In some embodiments, a targeted therapy is determined not to
be effective (e.g., the targeted therapy has poor efficacy) when
the cell-free tumor load (cfTL) identified at the second time point
is not substantially decreased (e.g., less than about 10%, less
than about 9%, less than about 8%, less than about 7%, less than
about 6%, less than about 5%, less than about 4%, less than about
3%, less than about 2%, less than about 1%, less than about 0.5%,
less than about 0.25%, less than about 0.2%, less than about 0.1%,
less than about 0.05%, less than about 0.01%) as compared to the
amount of the cfTL identified at the first time point.
[0087] In some embodiments, methods provided herein for determining
the efficacy of a targeted therapy include detecting the level of
at least one genetic alteration of circulating tumor DNA (ctDNA)
present in cell-free DNA, where the cell-free DNA is present in an
amount less than about 1500 ng, e.g., less than about 1400 ng, less
than about 1300 ng, less than about 1200 ng, less than about 1100
ng, less than about 1000 ng, less than about 900 ng, less than
about 800 ng, less than about 700 ng, less than about 600 ng, less
than about 500 ng, less than about 400 ng, less than about 300 ng,
less than about 200 ng, less than about 150 ng, less than about 100
ng, less than about 95 ng, less than about 90 ng, less than about
85 ng, less than about 80 ng, less than about 75 ng, less than
about 70 ng, less than about 65 ng, less than about 60 ng, less
than about 55 ng, less than about 50 ng, less than about 45 ng,
less than about 40 ng, less than about 35 ng, less than about 30
ng, less than about 25 ng, less than about 20 ng, less than about
15 ng, less than about 10 ng, or less than about 5 ng.
[0088] In some embodiments, after determining the efficacy of a
targeted therapy administered to a subject, the subject can be
administered a diagnostic test (e.g., any of the diagnostic tests
disclosed herein) and/or monitored (e.g., according to any of the
monitoring methods, schedules, etc. disclosed herein). In some
embodiments, after determining the efficacy of a targeted therapy
administered to a subject, the subject can be selected for further
diagnostic testing (e.g., using any of the diagnostic tests
disclosed herein) and/or selected for increased monitoring (e.g.,
according to any of the increased monitoring methods, schedules,
etc. disclosed herein). For example, a subject can be administered
a targeted therapy, and the targeted therapy is determined to be
effective, and the subject can then be administered a diagnostic
test and/or selected for further diagnostic testing (e.g., to
confirm the effectiveness of the targeted therapy). As another
example, a subject can be administered a targeted therapy, where
was determined to be effective, and the subject can then be
monitored and/or selected for increased monitoring (e.g., to keep
watch for the reemergence of the same or another cancer).
[0089] In some embodiments, a targeted therapy is determined to be
effective in a subject. In such embodiments, the subject may be
administered one or more additional doses of the effective targeted
therapy during the course of treatment. In some embodiments, when a
targeted therapy is determined to be effective in a subject, the
subject may be administered one or more additional doses of the
effective targeted therapy during the course of treatment without
being administered other therapeutic interventions (e.g. other
therapeutic interventions to treat the same condition the targeted
therapy treats, e.g., cancer). In some embodiments, when a targeted
therapy is determined to be effective in a subject, the subject may
be administered one or more additional doses of the effective
targeted therapy, and may further be administered one or more
therapeutic interventions (e.g., any of the therapeutic
interventions disclosed herein) during the course of treatment.
[0090] In some embodiments, a targeted therapy is determined not to
be effective in a subject (e.g., the targeted therapy has poor
efficacy). In such embodiments, the subject may be administered a
therapeutic intervention (e.g., any of the therapeutic
interventions disclosed herein) that is different that the
ineffective targeted therapy (e.g., a different class of targeted
therapy or a different targeted therapy within the same type of
targeted therapy that was determined to be ineffective) during the
course of treatment. As non-limiting examples, a subject may be
administered a different a targeted therapy, a chemotherapy,
immunotherapy, radiation therapy, and/or surgery. Those of ordinary
skill in the art will be aware of suitable therapeutic
interventions to administer when the targeted therapy is determined
not to be effective.
[0091] In some aspects, methods provided herein include obtaining
from the subject additional sample(s) at additional time point(s)
(e.g., at a third time point, a fourth time point, etc.) and
determining the efficacy of a targeted therapy at the additional
time point(s).
[0092] In some aspects, the second time point is about one to about
four weeks (e.g., about one to about three weeks, about one to
about two weeks, about two to about four weeks, about two to about
three weeks, about three weeks to about four weeks; about 2 days to
about 30 days, about 2 days to about 28 days, about 2 days to about
26 days, about 2 days to about 24 days, about 2 days to about 22
days, about 2 days to about 20 days, about 2 days to about 18 days,
about 2 days to about 16 days, about 2 days to about 14 days, about
2 days to about 12 days, about 2 days to about 10 days, about 2
days to about 8 days, about 2 days to about 6 days, about 2 days to
about 4 days, about 4 days to about 30, about 4 days to about 28
days, about 4 days to about 26 days, about 4 days to about 24 days,
about 4 days to about 22 days, about 4 days to about 20 days, about
4 days to about 18 days, about 4 days to about 16 days, about 4
days to about 14 days, about 4 days to about 12 days, about 4 days
to about 10 days, about 4 days to about 8 days, about 4 days to
about 6 days, about 6 days to about 30, about 6 days to about 28
days, about 6 days to about 26 days, about 6 days to about 24 days,
about 6 days to about 22 days, about 6 days to about 20 days, about
6 days to about 18 days, about 6 days to about 16 days, about 6
days to about 14 days, about 6 days to about 12 days, about 6 days
to about 10 days, about 6 days to about 8 days, about 8 days to
about 30, about 8 days to about 28 days, about 8 days to about 26
days, about 8 days to about 24 days, about 8 days to about 22 days,
about 8 days to about 20 days, about 8 days to about 18 days, about
8 days to about 16 days, about 8 days to about 14 days, about 8
days to about 12 days, about 8 days to about 10 days, about 10 days
to about 30, about 10 days to about 28 days, about 10 days to about
26 days, about 10 days to about 24 days, about 10 days to about 22
days, about 10 days to about 20 days, about 10 days to about 18
days, about 10 days to about 16 days, about 10 days to about 14
days, about 10 days to about 12 days, about 12 days to about 30,
about 12 days to about 28 days, about 12 days to about 26 days,
about 12 days to about 24 days, about 12 days to about 22 days,
about 12 days to about 20 days, about 12 days to about 18 days,
about 12 days to about 16 days, about 12 days to about 14 days,
about 14 days to about 30, about 14 days to about 28 days, about 14
days to about 26 days, about 14 days to about 24 days, about 14
days to about 22 days, about 14 days to about 20 days, about 14
days to about 18 days, about 14 days to about 16 days, about 16
days to about 30, about 16 days to about 28 days, about 16 days to
about 26 days, about 16 days to about 24 days, about 16 days to
about 22 days, about 16 days to about 20 days, about 16 days to
about 18 days, about 18 days to about 30, about 18 days to about 28
days, about 18 days to about 26 days, about 18 days to about 24
days, about 18 days to about 22 days, about 18 days to about 20
days, about 20 days to about 30, about 20 days to about 28 days,
about 20 days to about 26 days, about 20 days to about 24 days,
about 20 days to about 22 days, about 22 days to about 30, about 22
days to about 28 days, about 22 days to about 26 days, about 22
days to about 24 days, about 24 days to about 30, about 24 days to
about 28 days, about 24 days to about 26 days, about 26 days to
about 30, about 26 days to about 28 days, about 26 days to about
30; about 1 day, about 2 days, about 4 days, about 6 days, about 8
days, about 10 days, about 12 days, about 14 days, about 16 days,
about 18 days, about 20 days, about 22 days, about 24 days, about
26 days, about 28 days, or about 30 days) after the first time
point.
Determining, Monitoring, and Treating Resistance to a Targeted
Therapy
[0093] Also provided herein are methods for determining that a
subject that has developed resistance to a targeted therapy (e.g.,
any of targeted therapy disclosed herein or known in the art),
methods for monitoring a subject for the development of resistance
to a targeted therapy, and methods for treating such subjects with
a different therapeutic intervention.
[0094] Provided herein are methods of determining acquired
resistance to a targeted therapy in a subject having cancer that
include: detecting a first cell-free tumor load (cfTL) in a
biological sample isolated from the subject at a first time point;
detecting a second cfTL in a biological sample obtained from the
subject at a second time point, wherein the subject has received at
least one dose of the targeted therapy between the first time point
and the second time point; and identifying as having acquired
resistance when the second cfTL is not substantially reduced as
compared to the first cfTL. In some embodiments, the subject
determined to have developed resistance to the targeted therapy
exhibits a decreased level of at least one genetic alteration of
ctDNA at a time point between the first and second time points
(e.g., the level of the genetic alteration of ctDNA initially
decreases upon administration of the targeted therapy, but then
increases when the subject develops resistance).
[0095] Provided herein are methods of determining acquired
resistance to a targeted therapy in a subject having cancer that
include: detecting a first level of at least one genetic alteration
in circulating tumor DNA (ctDNA) in a biological sample isolated
from the subject at a first time point; detecting a second level of
the at least one genetic alteration in circulating tumor DNA
(ctDNA) in a biological sample obtained from the subject at a
second time point, wherein the subject has received at least one
dose of the targeted therapy between the first time point and the
second time point; and identifying the subject as having acquired
resistance when the second level of the at least one genetic
alteration is substantially increased as compared to the first
level of the at least one genetic alteration.
[0096] Provided herein are methods of determining acquired
resistance to a targeted therapy in a subject having cancer that
include: detecting a first level of aneuploidy in a biological
sample isolated from the subject at a first time point; detecting a
second level of aneuploidy in a biological sample obtained from the
subject at a second time point, wherein the subject has received at
least one dose of the targeted therapy between the first time point
and the second time point; and identifying the subject as having
acquired resistance when the second level of aneuploidy is
substantially increased as compared to the first level of
aneuploidy.
[0097] In some embodiments, a subject is determined not to have
developed resistance to a targeted therapy when the amount of at
least one genetic alteration of circulating tumor DNA (ctDNA)
identified at the second time point is decreased by at least about
2-fold, at least about 3-fold, at least about 4-fold, at least
about 5-fold, at least about 6-fold, at least about 7-fold, at
least about 8-fold, at least about 9-fold, at least about 10-fold
or more compared to the amount of the at least one genetic
alteration of circulating tumor DNA (ctDNA) identified at the first
time point. In some embodiments, a subject is determined not to
have developed resistance to a targeted therapy when the amount of
at least one genetic alteration of circulating tumor DNA (ctDNA)
identified at the second time point is decreased by at least about
20%, at least about 25%, at least about 30%, at least about 35%, at
least about 40%, at least about 45%, at least about 50%, at least
about 55%, at least about 60%, at least about 65%, at least about
70%, at least about 75%, at least about 80%, at least about 85%, at
least about 90%, at least about 95%, at least about 96%, at least
about 97%, at least about 98%, at least about 99% or more compared
to the amount of the at least one genetic alteration of circulating
tumor DNA (ctDNA) identified at the first time point. In some
embodiments, a subject is determined not to have developed
resistance to a targeted therapy when circulating tumor DNA (ctDNA)
is not observed at the second time point.
[0098] In some embodiments, provided herein are methods of
determining that a subject has not developed resistance to a
targeted therapy (e.g., any of the targeted therapies disclosed
herein or known in the art), including: detecting a first cell-free
tumor load (cfTL) in a biological sample isolated from the subject
at a first time point; detecting a second cfTL in a biological
sample obtained from the subject at a second time point, wherein
the subject has received at least one dose of the targeted therapy
between the first time point and the second time point; and
identifying the subject as having not acquired resistance when the
second cfTL is reduced as compared to the first cfTL.
[0099] In some embodiments, provided herein are methods of
determining that a subject has developed resistance to a targeted
therapy (e.g., any of the targeted therapies disclosed herein or
known in the art), including: detecting a first cell-free tumor
load (cfTL) in a biological sample isolated from the subject at a
first time point; detecting a second cfTL in a biological sample
obtained from the subject at a second time point, wherein the
subject has received at least one dose of the targeted therapy
between the first time point and the second time point; and
identifying the subject as having acquired resistance when the
second cfTL is not substantially reduced as compared to the first
cfTL.
[0100] In some embodiments, the subject determined to have
developed resistance to the targeted therapy exhibits an increased
cell-free tumor load (cfTL) at a time point between the first and
second time points (e.g., cell-free tumor load (cfTL) initially
increases upon administration of the targeted therapy, but then
decreases when the subject develops resistance).
[0101] In some embodiments, detecting and comparing cfTL levels at
different time points results in a more rapid determination of
whether the subject has developed resistance than conventional
methods (e.g., imaging or scanning).
[0102] In some embodiments, a subject is identified as having
developed resistance to an administered targeted therapy when the
second cfTL not substantially reduced as compared to the first cfTL
(e.g., an increase in second cfTL of less than about 10%, less than
about 9%, less than about 8%, less than about 7%, less than about
6%, less than about 5%, less than about 4%, less than about 3%,
less than about 2%, less than about 1%, less than about 0.5%, less
than about 0.25%, less than about 0.2%, less than about 0.1%, less
than about 0.05%, or less than about 0.01% as compared to the first
cfTL).
[0103] In some embodiments, methods of determining that a subject
that has developed resistance to a targeted therapy (e.g., any of
targeted therapy disclosed herein or known in the art) include
using any of the methods disclosed herein for detecting the
presence or level of at least one genetic alteration of circulating
tumor DNA (ctDNA).
[0104] In some embodiments, a subject is determined to have
developed resistance to a targeted therapy when that targeted
therapy is no longer effective or is less effective than it was
when first administered. For example, a subject can be determined
to have developed resistance to a targeted therapy when the
targeted therapy is at least 20%, 25%, 30%, 35%, 40% 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or any percentage
within between, less effective than when the targeted therapy was
first administered. The effectiveness of a targeted therapy, both
when it is first administered and during the course of the
treatment, can be determined by any of a variety of methods and
techniques. For example, the size and/or position of the tumor (as
determined, e.g., by scanning or imaging technologies), the number
of cancer cells, the amount of cell-free DNA, and/or the amount of
genetic alterations of circulating tumor DNA can be determined and
used to assess whether a subject has developed resistance to the
targeted therapy. Other suitable methods and techniques are known
in the art. In some embodiments, after determining that a subject
that has developed resistance to a targeted therapy, a different
targeted therapy and/or therapeutic intervention (e.g., any of the
therapeutic interventions disclosed herein or known in the art) is
selected and/or administered to the subject.
[0105] In some embodiments, methods for monitoring a subject for
the development of resistance to a targeted therapy (e.g., any of
targeted therapy disclosed herein or known in the art) include
using any of the methods disclosed herein for detecting the
presence or level of at least one genetic alteration of circulating
tumor DNA (ctDNA).
[0106] In some embodiments, methods for treating a subject that has
developed resistance to a therapeutic intervention (e.g., any of
the therapeutic interventions disclosed herein or known in the art)
include using any of the methods disclosed herein for detecting at
least one genetic alteration of circulating tumor DNA.
[0107] In some embodiments methods provided herein for determining
that a subject that has developed resistance to a targeted therapy,
for monitoring a subject for the development of resistance to a
targeted therapy, and/or for treating such subjects with a
different therapeutic intervention include determining the level of
at least one genetic alteration of circulating tumor DNA present in
cell-free DNA, where the circulating tumor DNA represents 100% of
the cell-free DNA.
[0108] In some aspects, methods provided herein include obtaining
from the subject additional sample(s) at additional time point(s)
(e.g., at a third time point, a fourth time point, etc.) and
determining whether a subject has developed resistance to a
targeted therapy at the additional time point(s).
[0109] In some aspects, the second time point is about one to about
four weeks (e.g., about one to about three weeks, about one to
about two weeks, about two to about four weeks, about two to about
three weeks, about three weeks to about four weeks; about 2 days to
about 30 days, about 2 days to about 28 days, about 2 days to about
26 days, about 2 days to about 24 days, about 2 days to about 22
days, about 2 days to about 20 days, about 2 days to about 18 days,
about 2 days to about 16 days, about 2 days to about 14 days, about
2 days to about 12 days, about 2 days to about 10 days, about 2
days to about 8 days, about 2 days to about 6 days, about 2 days to
about 4 days, about 4 days to about 30, about 4 days to about 28
days, about 4 days to about 26 days, about 4 days to about 24 days,
about 4 days to about 22 days, about 4 days to about 20 days, about
4 days to about 18 days, about 4 days to about 16 days, about 4
days to about 14 days, about 4 days to about 12 days, about 4 days
to about 10 days, about 4 days to about 8 days, about 4 days to
about 6 days, about 6 days to about 30, about 6 days to about 28
days, about 6 days to about 26 days, about 6 days to about 24 days,
about 6 days to about 22 days, about 6 days to about 20 days, about
6 days to about 18 days, about 6 days to about 16 days, about 6
days to about 14 days, about 6 days to about 12 days, about 6 days
to about 10 days, about 6 days to about 8 days, about 8 days to
about 30, about 8 days to about 28 days, about 8 days to about 26
days, about 8 days to about 24 days, about 8 days to about 22 days,
about 8 days to about 20 days, about 8 days to about 18 days, about
8 days to about 16 days, about 8 days to about 14 days, about 8
days to about 12 days, about 8 days to about 10 days, about 10 days
to about 30, about 10 days to about 28 days, about 10 days to about
26 days, about 10 days to about 24 days, about 10 days to about 22
days, about 10 days to about 20 days, about 10 days to about 18
days, about 10 days to about 16 days, about 10 days to about 14
days, about 10 days to about 12 days, about 12 days to about 30,
about 12 days to about 28 days, about 12 days to about 26 days,
about 12 days to about 24 days, about 12 days to about 22 days,
about 12 days to about 20 days, about 12 days to about 18 days,
about 12 days to about 16 days, about 12 days to about 14 days,
about 14 days to about 30, about 14 days to about 28 days, about 14
days to about 26 days, about 14 days to about 24 days, about 14
days to about 22 days, about 14 days to about 20 days, about 14
days to about 18 days, about 14 days to about 16 days, about 16
days to about 30, about 16 days to about 28 days, about 16 days to
about 26 days, about 16 days to about 24 days, about 16 days to
about 22 days, about 16 days to about 20 days, about 16 days to
about 18 days, about 18 days to about 30, about 18 days to about 28
days, about 18 days to about 26 days, about 18 days to about 24
days, about 18 days to about 22 days, about 18 days to about 20
days, about 20 days to about 30, about 20 days to about 28 days,
about 20 days to about 26 days, about 20 days to about 24 days,
about 20 days to about 22 days, about 22 days to about 30, about 22
days to about 28 days, about 22 days to about 26 days, about 22
days to about 24 days, about 24 days to about 30, about 24 days to
about 28 days, about 24 days to about 26 days, about 26 days to
about 30, about 26 days to about 28 days, about 26 days to about
30; about 1 day, about 2 days, about 4 days, about 6 days, about 8
days, about 10 days, about 12 days, about 14 days, about 16 days,
about 18 days, about 20 days, about 22 days, about 24 days, about
26 days, about 28 days, or about 30 days) after the first time
point.
[0110] In some aspects, the second time point is about 1 hour to
about 7 days (e.g., about 1 hour to about 6 days, about 1 hour to
about 5 days, about 1 hour to about 4 days, about 1 hour to about
72 hours, about 1 hour to about 66 hours, about 1 hour to about 60
hours, about 1 hour to about 54 hours, about 1 hour to about 48
hours, about 1 hour to about 42 hours, about 1 hour to about 36
hours, about 1 hour to about 30 hours, about 1 hour to about 24
hours, about 1 hour to about 18 hours, about 1 hour to about 12
hours, about 1 hour to about 6 hours, about 1 hour to about 4
hours, about 1 hour to about 2 hours, about 2 hours to about 7
days, about 2 hours to about 6 days, about 2 hours to about 5 days,
about 2 hours to about 4 days, about 2 hours to about 72 hours,
about 2 hours to about 66 hours, about 2 hours to about 60 hours,
about 2 hours to about 54 hours, about 2 hours to about 48 hours,
about 2 hours to about 42 hours, about 2 hours to about 36 hours,
about 2 hours to about 30 hours, about 2 hours to about 24 hours,
about 2 hours to about 18 hours, about 2 hours to about 12 hours,
about 2 hours to about 6 hours, about 2 hours to about 4 hours,
about 4 hours to about 7 days, about 4 hours to about 6 days, about
4 hours to about 5 days, about 4 hours to about 4 days, about 4
hours to about 72 hours, about 4 hours to about 66 hours, about 4
hours to about 60 hours, about 4 hours to about 54 hours, about 4
hours to about 48 hours, about 4 hours to about 42 hours, about 4
hours to about 36 hours, about 4 hours to about 30 hours, about 4
hours to about 24 hours, about 4 hours to about 18 hours, about 4
hours to about 12 hours, about 4 hours to about 6 hours, about 6
hours to about 7 days, about 6 hours to about 6 days, about 6 hours
to about 5 days, about 6 hours to about 4 days, about 6 hours to
about 72 hours, about 6 hours to about 66 hours, about 6 hours to
about 60 hours, about 6 hours to about 54 hours, about 6 hours to
about 48 hours, about 6 hours to about 42 hours, about 6 hours to
about 36 hours, about 6 hours to about 30 hours, about 6 hours to
about 24 hours, about 6 hours to about 18 hours, about 6 hours to
about 12 hours, about 12 hours to about 7 days, about 12 hours to
about 6 days, about 12 hours to about 5 days, about 12 hours to
about 4 days, about 12 hours to about 72 hours, about 12 hours to
about 66 hours, about 12 hours to about 60 hours, about 12 hours to
about 54 hours, about 12 hours to about 48 hours, about 12 hours to
about 42 hours, about 12 hours to about 36 hours, about 12 hours to
about 30 hours, about 12 hours to about 24 hours, about 12 hours to
about 18 hours, about 18 hours to about 7 days, about 18 hours to
about 6 days, about 18 hours to about 5 days, about 18 hours to
about 4 days, about 18 hours to about 72 hours, about 18 hours to
about 66 hours, about 18 hours to about 60 hours, about 18 hours to
about 54 hours, about 18 hours to about 48 hours, about 18 hours to
about 42 hours, about 18 hours to about 36 hours, about 18 hours to
about 30 hours, about 18 hours to about 24 hours, about 24 hours to
about 7 days, about 24 hours to about 6 days, about 24 hours to
about 5 days, about 24 hours to about 4 days, about 24 hours to
about 72 hours, about 24 hours to about 66 hours, about 24 hours to
about 60 hours, about 24 hours to about 54 hours, about 24 hours to
about 48 hours, about 24 hours to about 42 hours, about 24 hours to
about 36 hours, about 24 hours to about 30 hours, about 30 hours to
about 7 days, about 30 hours to about 6 days, about 30 hours to
about 5 days, about 30 hours to about 4 days, about 30 hours to
about 72 hours, about 30 hours to about 66 hours, about 30 hours to
about 60 hours, about 30 hours to about 54 hours, about 30 hours to
about 48 hours, about 30 hours to about 42 hours, about 30 hours to
about 36 hours, about 36 hours to about 7 days, about 36 hours to
about 6 days, about 36 hours to about 5 days, about 36 hours to
about 4 days, about 36 hours to about 72 hours, about 36 hours to
about 66 hours, about 36 hours to about 60 hours, about 36 hours to
about 54 hours, about 36 hours to about 48 hours, about 36 hours to
about 42 hours, about 42 hours to about 7 days, about 42 hours to
about 6 days, about 42 hours to about 5 days, about 42 hours to
about 4 days, about 42 hours to about 72 hours, about 42 hours to
about 66 hours, about 42 hours to about 60 hours, about 42 hours to
about 54 hours, about 42 hours to about 48 hours, about 48 hours to
about 7 days, about 48 hours to about 6 days, about 48 hours to
about 5 days, about 48 hours to about 4 days, about 48 hours to
about 72 hours, about 48 hours to about 66 hours, about 48 hours to
about 60 hours, about 48 hours to about 54 hours, about 54 hours to
about 7 days, about 54 hours to about 6 days, about 54 hours to
about 5 days, about 54 hours to about 4 days, about 54 hours to
about 72 hours, about 54 hours to about 66 hours, about 54 hours to
about 60 hours, about 60 hours to about 7 days, about 60 hours to
about 6 days, about 60 hours to about 5 days, about 60 hours to
about 4 days, about 60 hours to about 72 hours, about 60 hours to
about 66 hours, about 66 hours to about 7 days, about 66 hours to
about 6 days, about 66 hours to about 5 days, about 66 hours to
about 4 days, about 66 hours to about 72 hours, about 72 hours to
about 7 days, about 72 hours to about 6 days, about 72 hours to
about 5 days, about 72 hours to about 4 days, about 4 days to about
7 days, about 4 days to about 6 days, about 4 days to about 5 days,
about 5 days to about 7 days, about 5 days to about 6 days, about 6
days to about 7 days; about 1 hour, about 2 hours, about 4 hours,
about 6 hours, about 8 hours, about 10 hours, about 12 hours, about
18 hours, about 24 hours, about 30 hours, about 36 hours, about 42
hours, about 48 hours, about 54 hours, about 60 hours, about 66
hours, about 72 hours, about 4 days, about 5 days, about 6 days, or
about 7 days) after the first time point.
Determining Cell-Free Tumor Load in a Subject
[0111] Also provided herein are methods for determining cell-free
tumor load (cfTL). In some embodiments, cfTL is detected in a
biological sample isolated from the subject at a first time point.
In some embodiments, cfTL is detected in a biological sample
isolated from the subject at a second time point. In some
embodiments, the subject has received at least one dose of the
targeted therapy between the first time point and the second time
point.
[0112] In some embodiments, determining cell-free tumor load (cfTL)
in a subject includes detecting a first level of at least one
genetic alteration present in ctDNA and/or a first level of
aneuploidy in a biological sample isolated from the subject at a
first time point. In some embodiments, determining cell-free tumor
load (cfTL) in a subject includes detecting a first level of at
least one genetic alteration present in ctDNA and/or a first level
of aneuploidy in a biological sample isolated from the subject at a
second time point. In some embodiments, the subject has received at
least one dose of the targeted therapy between the first time point
and the second time point.
[0113] In some embodiments, determining cell-free tumor load (cfTL)
in a subject includes detecting the level of at least one genetic
alteration (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
genetic alterations) present in ctDNA present in a biological
sample isolated from the subject. In some embodiments, no genetic
alterations are detected in the subject, and determining cell-free
tumor load (cfTL) in a subject includes detecting the level of
aneuploidy in the subject.
[0114] In some embodiments, the subject exhibits a cfTL that is
reduced at a second time point as compared to a first time point.
In some embodiments, the subject exhibits a cfTL that is not
reduced at a second time point as compared to a first time
point.
[0115] In some embodiments, the second cfTL is not substantially
reduced as compared to the first cfTL (e.g., an increase in second
cfTL of less than about 10%, less than about 9%, less than about
8%, less than about 7%, less than about 6%, less than about 5%,
less than about 4%, less than about 3%, less than about 2%, less
than about 1%, less than about 0.5%, less than about 0.25%, less
than about 0.2%, less than about 0.1%, less than about 0.05%, or
less than about 0.01% as compared to the first cfTL, or a decrease
of at least about 0.5%, at least about 1%, at least about 2%, at
least about 4%, at least about 6%, at least about 8%, at least
about 10%, at least about 12%, at least about 14%, at least about
16%, at least about 18%, at least about 20%, at least about 22%, at
least about 24%, at least about 26%, at least about 28%, at least
about 30%, at least about 40%, at least about 45%, at least about
50%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, or at least about 99% in the second
cfTL as compared to the first cfTL.
[0116] In some aspects, methods provided herein include obtaining
from the subject additional sample(s) at additional time point(s)
(e.g., at a third time point, a fourth time point, etc.) and
determining whether a subject has developed resistance to a
targeted therapy at the additional time point(s).
[0117] In some aspects, the second time point is about one to about
four weeks (e.g., about one to about three weeks, about one to
about two weeks, about two to about four weeks, about two to about
three weeks, about three weeks to about four weeks; about 2 days to
about 30 days, about 2 days to about 28 days, about 2 days to about
26 days, about 2 days to about 24 days, about 2 days to about 22
days, about 2 days to about 20 days, about 2 days to about 18 days,
about 2 days to about 16 days, about 2 days to about 14 days, about
2 days to about 12 days, about 2 days to about 10 days, about 2
days to about 8 days, about 2 days to about 6 days, about 2 days to
about 4 days, about 4 days to about 30, about 4 days to about 28
days, about 4 days to about 26 days, about 4 days to about 24 days,
about 4 days to about 22 days, about 4 days to about 20 days, about
4 days to about 18 days, about 4 days to about 16 days, about 4
days to about 14 days, about 4 days to about 12 days, about 4 days
to about 10 days, about 4 days to about 8 days, about 4 days to
about 6 days, about 6 days to about 30, about 6 days to about 28
days, about 6 days to about 26 days, about 6 days to about 24 days,
about 6 days to about 22 days, about 6 days to about 20 days, about
6 days to about 18 days, about 6 days to about 16 days, about 6
days to about 14 days, about 6 days to about 12 days, about 6 days
to about 10 days, about 6 days to about 8 days, about 8 days to
about 30, about 8 days to about 28 days, about 8 days to about 26
days, about 8 days to about 24 days, about 8 days to about 22 days,
about 8 days to about 20 days, about 8 days to about 18 days, about
8 days to about 16 days, about 8 days to about 14 days, about 8
days to about 12 days, about 8 days to about 10 days, about 10 days
to about 30, about 10 days to about 28 days, about 10 days to about
26 days, about 10 days to about 24 days, about 10 days to about 22
days, about 10 days to about 20 days, about 10 days to about 18
days, about 10 days to about 16 days, about 10 days to about 14
days, about 10 days to about 12 days, about 12 days to about 30,
about 12 days to about 28 days, about 12 days to about 26 days,
about 12 days to about 24 days, about 12 days to about 22 days,
about 12 days to about 20 days, about 12 days to about 18 days,
about 12 days to about 16 days, about 12 days to about 14 days,
about 14 days to about 30, about 14 days to about 28 days, about 14
days to about 26 days, about 14 days to about 24 days, about 14
days to about 22 days, about 14 days to about 20 days, about 14
days to about 18 days, about 14 days to about 16 days, about 16
days to about 30, about 16 days to about 28 days, about 16 days to
about 26 days, about 16 days to about 24 days, about 16 days to
about 22 days, about 16 days to about 20 days, about 16 days to
about 18 days, about 18 days to about 30, about 18 days to about 28
days, about 18 days to about 26 days, about 18 days to about 24
days, about 18 days to about 22 days, about 18 days to about 20
days, about 20 days to about 30, about 20 days to about 28 days,
about 20 days to about 26 days, about 20 days to about 24 days,
about 20 days to about 22 days, about 22 days to about 30, about 22
days to about 28 days, about 22 days to about 26 days, about 22
days to about 24 days, about 24 days to about 30, about 24 days to
about 28 days, about 24 days to about 26 days, about 26 days to
about 30, about 26 days to about 28 days, about 26 days to about
30; about 1 day, about 2 days, about 4 days, about 6 days, about 8
days, about 10 days, about 12 days, about 14 days, about 16 days,
about 18 days, about 20 days, about 22 days, about 24 days, about
26 days, about 28 days, or about 30 days) after the first time
point.
[0118] In some aspects, the second time point is about 1 hour to
about 7 days (e.g., about 1 hour to about 6 days, about 1 hour to
about 5 days, about 1 hour to about 4 days, about 1 hour to about
72 hours, about 1 hour to about 66 hours, about 1 hour to about 60
hours, about 1 hour to about 54 hours, about 1 hour to about 48
hours, about 1 hour to about 42 hours, about 1 hour to about 36
hours, about 1 hour to about 30 hours, about 1 hour to about 24
hours, about 1 hour to about 18 hours, about 1 hour to about 12
hours, about 1 hour to about 6 hours, about 1 hour to about 4
hours, about 1 hour to about 2 hours, about 2 hours to about 7
days, about 2 hours to about 6 days, about 2 hours to about 5 days,
about 2 hours to about 4 days, about 2 hours to about 72 hours,
about 2 hours to about 66 hours, about 2 hours to about 60 hours,
about 2 hours to about 54 hours, about 2 hours to about 48 hours,
about 2 hours to about 42 hours, about 2 hours to about 36 hours,
about 2 hours to about 30 hours, about 2 hours to about 24 hours,
about 2 hours to about 18 hours, about 2 hours to about 12 hours,
about 2 hours to about 6 hours, about 2 hours to about 4 hours,
about 4 hours to about 7 days, about 4 hours to about 6 days, about
4 hours to about 5 days, about 4 hours to about 4 days, about 4
hours to about 72 hours, about 4 hours to about 66 hours, about 4
hours to about 60 hours, about 4 hours to about 54 hours, about 4
hours to about 48 hours, about 4 hours to about 42 hours, about 4
hours to about 36 hours, about 4 hours to about 30 hours, about 4
hours to about 24 hours, about 4 hours to about 18 hours, about 4
hours to about 12 hours, about 4 hours to about 6 hours, about 6
hours to about 7 days, about 6 hours to about 6 days, about 6 hours
to about 5 days, about 6 hours to about 4 days, about 6 hours to
about 72 hours, about 6 hours to about 66 hours, about 6 hours to
about 60 hours, about 6 hours to about 54 hours, about 6 hours to
about 48 hours, about 6 hours to about 42 hours, about 6 hours to
about 36 hours, about 6 hours to about 30 hours, about 6 hours to
about 24 hours, about 6 hours to about 18 hours, about 6 hours to
about 12 hours, about 12 hours to about 7 days, about 12 hours to
about 6 days, about 12 hours to about 5 days, about 12 hours to
about 4 days, about 12 hours to about 72 hours, about 12 hours to
about 66 hours, about 12 hours to about 60 hours, about 12 hours to
about 54 hours, about 12 hours to about 48 hours, about 12 hours to
about 42 hours, about 12 hours to about 36 hours, about 12 hours to
about 30 hours, about 12 hours to about 24 hours, about 12 hours to
about 18 hours, about 18 hours to about 7 days, about 18 hours to
about 6 days, about 18 hours to about 5 days, about 18 hours to
about 4 days, about 18 hours to about 72 hours, about 18 hours to
about 66 hours, about 18 hours to about 60 hours, about 18 hours to
about 54 hours, about 18 hours to about 48 hours, about 18 hours to
about 42 hours, about 18 hours to about 36 hours, about 18 hours to
about 30 hours, about 18 hours to about 24 hours, about 24 hours to
about 7 days, about 24 hours to about 6 days, about 24 hours to
about 5 days, about 24 hours to about 4 days, about 24 hours to
about 72 hours, about 24 hours to about 66 hours, about 24 hours to
about 60 hours, about 24 hours to about 54 hours, about 24 hours to
about 48 hours, about 24 hours to about 42 hours, about 24 hours to
about 36 hours, about 24 hours to about 30 hours, about 30 hours to
about 7 days, about 30 hours to about 6 days, about 30 hours to
about 5 days, about 30 hours to about 4 days, about 30 hours to
about 72 hours, about 30 hours to about 66 hours, about 30 hours to
about 60 hours, about 30 hours to about 54 hours, about 30 hours to
about 48 hours, about 30 hours to about 42 hours, about 30 hours to
about 36 hours, about 36 hours to about 7 days, about 36 hours to
about 6 days, about 36 hours to about 5 days, about 36 hours to
about 4 days, about 36 hours to about 72 hours, about 36 hours to
about 66 hours, about 36 hours to about 60 hours, about 36 hours to
about 54 hours, about 36 hours to about 48 hours, about 36 hours to
about 42 hours, about 42 hours to about 7 days, about 42 hours to
about 6 days, about 42 hours to about 5 days, about 42 hours to
about 4 days, about 42 hours to about 72 hours, about 42 hours to
about 66 hours, about 42 hours to about 60 hours, about 42 hours to
about 54 hours, about 42 hours to about 48 hours, about 48 hours to
about 7 days, about 48 hours to about 6 days, about 48 hours to
about 5 days, about 48 hours to about 4 days, about 48 hours to
about 72 hours, about 48 hours to about 66 hours, about 48 hours to
about 60 hours, about 48 hours to about 54 hours, about 54 hours to
about 7 days, about 54 hours to about 6 days, about 54 hours to
about 5 days, about 54 hours to about 4 days, about 54 hours to
about 72 hours, about 54 hours to about 66 hours, about 54 hours to
about 60 hours, about 60 hours to about 7 days, about 60 hours to
about 6 days, about 60 hours to about 5 days, about 60 hours to
about 4 days, about 60 hours to about 72 hours, about 60 hours to
about 66 hours, about 66 hours to about 7 days, about 66 hours to
about 6 days, about 66 hours to about 5 days, about 66 hours to
about 4 days, about 66 hours to about 72 hours, about 72 hours to
about 7 days, about 72 hours to about 6 days, about 72 hours to
about 5 days, about 72 hours to about 4 days, about 4 days to about
7 days, about 4 days to about 6 days, about 4 days to about 5 days,
about 5 days to about 7 days, about 5 days to about 6 days, about 6
days to about 7 days; about 1 hour, about 2 hours, about 4 hours,
about 6 hours, about 8 hours, about 10 hours, about 12 hours, about
18 hours, about 24 hours, about 30 hours, about 36 hours, about 42
hours, about 48 hours, about 54 hours, about 60 hours, about 66
hours, about 72 hours, about 4 days, about 5 days, about 6 days, or
about 7 days) after the first time point.
Identifying the Presence or Levels of Genetic Alterations in
Circulating Tumor DNA in a Subject
[0119] Provided herein are methods for identifying the presence or
level of at least one genetic alteration (e.g., at least two, at
least, three, at least four, at least five, at least six, at least
seven, at least eight, at least nine, at least ten, at least
eleven, at least twelve, at least thirteen, at least fourteen, at
least fifteen, at least sixteen, at least seventeen, at least
eighteen, at least nineteen, at least twenty, at least twenty-five,
at least thirty, at least thirty-five, at least forty, at least
forty-five, at least fifty, between 1 and 50, between 1 and 45,
between 1 and 40, between 1 and 35, between 1 and 30, between 1 and
25, between 1 and 20, between 1 and 15, between 1 and 10, between 1
and 5, between 5 and 10, between 5 and 15, between 5 and 20,
between 5 and 25, between 5 and 30, between 5 and 35, between 5 and
40, between 5 and 45, between 5 and 50, between 10 and 15, between
10 and 20, between 10 and 25, between 10 and 30, between 10 and 35,
between 10 and 40, between 10 and 45, between 10 and 50, between 15
and 20, between 15 and 25, between 15 and 30, between 15 and 35,
between 15 and 40, between 15 and 45, between 15 and 50, between 20
and 25, between 20 and 30, between 20 and 35, between 20 and 40,
between 20 and 50, between 25 and 30, between 25 and 50, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 genetic
alterations) of ctDNA in a subject (e.g., a first level of at least
one genetic alteration of ctDNA at a first time point and/or a
second level of at least one genetic alteration of ctDNA at a
second time point).
[0120] In some embodiments, the level of at least one genetic
alteration of ctDNA indicates the tumor burden in the subject.
[0121] In some embodiments, identifying the level of at least one
genetic alteration of ctDNA includes identifying the presence or
level of a mutation, a duplication, and/or substitution in a ctDNA
sequence. In some embodiments, identifying the level of at least
one genetic alteration of ctDNA includes identifying the presence
or level of aneuploidy of ctDNA.
[0122] In some embodiments, the biological sample is isolated from
subject. Any suitable biological sample that contains cell-free DNA
can be used in accordance with any of the variety of methods
disclosed herein. For example, the biological sample can include
blood, plasma, serum, urine, cerebrospinal fluid, saliva, sputum,
broncho-alveolar lavage, bile, lymphatic fluid, cyst fluid, stool,
uterine lavage, vaginal fluids, ascites, and combinations thereof.
Methods of isolating biological samples from a subject are known to
those of ordinary skill in the art.
[0123] In some embodiments, the step of detecting a genetic
alteration (e.g., one or more genetic alterations) in cell-free DNA
is performed using one or more of the methods described herein
(e.g., a targeted capture method (e.g., TEC-Seq), a next-generation
sequencing method, and an array-based method, or any combinations
thereof).
[0124] In some embodiments, methods provided herein can be used to
detect a genetic alteration (e.g., one or more genetic alterations)
in circulating tumor DNA present in cell-free DNA, where the
cell-free DNA is present in an amount less than about 1500 ng,
e.g., less than about 1400 ng, less than about 1300 ng, less than
about 1200 ng, less than about 1100 ng, less than about 1000 ng,
less than about 900 ng, less than about 800 ng, less than about 700
ng, less than about 600 ng, less than about 500 ng, less than about
400 ng, less than about 300 ng, less than about 200 ng, less than
about 150 ng, less than about 100 ng, less than about 95 ng, less
than about 90 ng, less than about 85 ng, less than about 80 ng,
less than about 75 ng, less than about 70 ng, less than about 65
ng, less than about 60 ng, less than about 55 ng, less than about
50 ng, less than about 45 ng, less than about 40 ng, less than
about 35 ng, less than about 30 ng, less than about 25 ng, less
than about 20 ng, less than about 15 ng, less than about 10 ng, or
less than about 5 ng. In some embodiments, methods provided herein
can be used to detect a genetic alteration (e.g., one or more
genetic alterations) in circulating tumor DNA present in cell-free
DNA, where the circulating tumor DNA represents 100% of the
cell-free DNA. In some embodiments, methods provided herein can be
used to detect a genetic alteration (e.g., one or more genetic
alterations) in circulating tumor DNA present in cell-free DNA,
where the circulating tumor DNA represents less than 100% of the
cell-free DNA, e.g. about 95%, about 90%, about 85%, about 80%,
about 75%, about 70%, about 65%, about 60%, about 55%, about 50%,
about 45%, about 40%, about 35%, about 30%, about 25%, about 20%,
about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, about
1%, about 0.95%, about 0.90%, about 0.85%, about 0.80%, about
0.75%, about 0.70%, about 0.65%, about 0.60%, about 0.55%, about
0.50%, about 0.45%, about 0.40%, about 0.35%, about 0.30%, about
0.25%, about 0.20%, about 0.15%, about 0.10%, about 0.09%, about
0.08%, about 0.07%, about 0.06%, about 0.05% of the cell-free DNA,
or less.
[0125] In some embodiments, a genetic alteration and/or aneuploidy
that is detected by any of the variety of methods disclosed herein
is present in a cancer cell present in the subject. For example, a
genetic alteration listed in Table 1 that is detected using any of
the variety of methods disclosed herein can be present in a cancer
cell present in the subject. In some embodiments, a genetic
alteration detected by any of the variety of methods disclosed
herein is confirmed to be present in a cancer cell present in the
subject through further diagnostic testing (e.g., diagnostic scans,
biopsies, molecular-based techniques to confirm the presence of the
cancer cell mutation, or any of the other diagnostic testing
methods disclosed herein or known in the art).
[0126] In some embodiments, detecting the presence or level of at
least one genetic alteration of ctDNA is performed using one or
more of the methods described herein (e.g., a targeted capture
method, a next-generation sequencing method, and an array-based
method, or any combinations thereof). In some embodiments,
detecting the presence or level of ctDNA is performed using
TEC-Seq, or a variation of TEC-Seq (Phallen et al., Science Transl
Med, (403), 2017). For example, detecting the presence or level of
at least one genetic alteration of ctDNA can include the following
steps: extracting cell-free DNA from blood, ligating a low
complexity pool of dual index barcode adapters to the cell-free DNA
to generate a plurality of barcode adapter-ligated cell-free DNA
segments, capturing the plurality of barcode adapter-ligated
cell-free DNA segments, sequencing the plurality of captured
barcode adapter-ligated cell-free DNA segments, aligning the
sequenced plurality of captured barcode adapter-ligated cell-free
DNA segments to a reference genome, and identifying sequence
alterations using aligned sequences of multiple distinct molecules
containing identical redundant changes. In some embodiments, the
presence or level of at least one genetic alteration of ctDNA is
detected (e.g., using a TEC-Seq approach) at two or more time
points (e.g., a first time point prior to administration of an
immunotherapy and a second time point after administration of the
immunotherapy). In some embodiments, an increase in the number or
level of sequence alterations (e.g., at a second time point)
indicates an increase in the level of ctDNA. In some embodiments,
an increase in the level of ctDNA indicates an increased tumor load
or tumor burden in the subject. In some embodiments, a decrease in
the number or level of sequence alterations (e.g., at a second time
point) indicates a decrease in the level of ctDNA. In some
embodiments, a decrease in the level of ctDNA indicates a decreased
tumor load or tumor burden in the subject.
[0127] In some embodiments, detecting the presence or level of at
least one genetic alteration of ctDNA is performed using sequencing
technology (e.g., a next-generation). A variety of sequencing
technologies are known in the art. For example, a variety of
technologies for detection and characterization of circulating
tumor DNA in cell-free DNA is described in Haber and Velculescu,
Blood-Based Analyses of Cancer: Circulating Tumor Cells and
Circulating Tumor DNA, Cancer Discov., June; 4(6):650-61. doi:
10.1158/2159-8290.CD-13-1014, 2014, incorporated herein by
reference in its entirety. Non-limiting examples of such techniques
include SafeSeqs (Kinde et. al, Detection and quantification of
rare mutations with massively parallel sequencing, Proc Natl Acad
Sci USA; 108, 9530-5, 2011), OnTarget (Forshew et al., Noninvasive
identification and monitoring of cancer mutations by targeted deep
sequencing of plasma DNA, Sci Transl Med; 4:136ra68, 2012), and
TamSeq (Thompson et al., Winnowing DNA for rare sequences: highly
specific sequence and methylation based enrichment. PLoS ONE,
7:e31597, 2012), each of which is incorporated herein by reference
in its entirety.
[0128] In some embodiments, detecting the presence or level of at
least one genetic alteration of ctDNA is performed using droplet
digital PCR (ddPCR). In some embodiments, detecting the presence or
level of at least one genetic alteration of ctDNA is performed
using other sequencing technologies, including but not limited to,
chain-termination techniques, shotgun techniques,
sequencing-by-synthesis methods, methods that utilize
microfluidics, other capture technologies, or any of the other
sequencing techniques known in the art that are useful for
detection of small amounts of DNA in a sample (e.g., circulating
tumor DNA in a cell-free DNA sample).
[0129] In some embodiments, detecting the presence or level of at
least one genetic alteration of ctDNA is performed using
array-based methods. For example, detecting the presence or level
of at least one genetic alteration of ctDNA can be performed using
a DNA microarray. In some embodiments, a DNA microarray can detect
the presence or level of at least one genetic alteration of ctDNA.
In some embodiments, cell-free DNA is amplified prior to detecting
the presence or level of at least one genetic alteration of ctDNA.
Non-limiting examples of array-based methods that can be used in
any of the methods described herein, include: a complementary DNA
(cDNA) microarray (Kumar et al. (2012) J. Pharm. Bioallied Sci.
4(1): 21-26; Laere et al. (2009) Methods Mol. Biol. 512: 71-98;
Mackay et al. (2003) Oncogene 22: 2680-2688; Alizadeh et al. (1996)
Nat. Genet. 14: 457-460), an oligonucleotide microarray (Kim et al.
(2006) Carcinogenesis 27(3): 392-404; Lodes et al. (2009) PLoS One
4(7): e6229), a bacterial artificial chromosome (BAC) clone chip
(Chung et al. (2004) Genome Res. 14(1): 188-196; Thomas et al.
(2005) Genome Res. 15(12): 1831-1837), a single-nucleotide
polymorphism (SNP) microarray (Mao et al. (2007) Curr. Genomics
8(4): 219-228; Jasmine et al. (2012) PLoS One 7(2): e31968), a
microarray-based comparative genomic hybridization array
(array-CGH) (Beers and Nederlof (2006) Breast Cancer Res. 8(3):
210; Pinkel et al. (2005) Nat. Genetics 37: S11-S17; Michels et al.
(2007) Genet. Med. 9: 574-584), a molecular inversion probe (MIP)
assay (Wang et al. (2012) Cancer Genet 205(7-8): 341-55; Lin et al.
(2010) BMC Genomics 11: 712). In some embodiments, the cDNA
microarray is an Affymetrix microarray (Irizarry (2003) Nucleic
Acids Res 31:e15; Dalma-Weiszhausz et al. (2006) Methods Enzymol.
410: 3-28), a NimbleGen microarray (Wei et al. (2008) Nucleic Acids
Res 36(9): 2926-2938; Albert et al. (2007) Nat. Methods 4:
903-905), an Agilent microarray (Hughes et al. (2001) Nat.
Biotechnol. 19(4): 342-347), or a BeadArray array (Liu et al.
(2017) Biosens Bioelectron 92: 596-601). In some embodiments, the
oligonucleotide microarray is a DNA tiling array (Mockler and Ecker
(2005) Genomics 85(1): 1-15; Bertone et al. (2006) Genome Res
16(2): 271-281). Other suitable array-based methods are known in
the art.
Selecting a Subject for Further Diagnostic Testing
[0130] Also provided herein are methods for selecting a subject for
further diagnostic testing. In some embodiments, methods for
selecting a subject for further diagnostic testing include
detecting cfTL (e.g., one or more genetic alterations and/or
aneuploidy) in cell-free DNA in a biological sample isolated from
the subject, and selecting a subject for further diagnostic testing
when identified sufficiently high cfTL is detected.
[0131] In some embodiments, the biological sample is isolated from
subject. Any suitable biological sample that contains cell-free DNA
can be used in accordance with any of the variety of methods
disclosed herein. For example, the biological sample can include
blood, plasma, serum, urine, cerebrospinal fluid, saliva, sputum,
broncho-alveolar lavage, bile, lymphatic fluid, cyst fluid, stool,
uterine lavage, vaginal fluids, ascites, and combinations thereof.
Methods of isolating biological samples from a subject are known to
those of ordinary skill in the art.
[0132] In some embodiments, the step of detecting a genetic
alteration (e.g., one or more genetic alterations) in cell-free DNA
is performed using one or more of the methods described herein
(e.g., a targeted capture method, a next-generation sequencing
method, and an array-based method, or any combinations
thereof).
[0133] In some embodiments, methods provided herein for selecting a
subject for further diagnostic testing include detecting cfTL
(e.g., one or more genetic alterations and/or aneuploidy) in
circulating tumor DNA present in cell-free DNA, where the cell-free
DNA is present in an amount less than about 1500 ng, e.g., less
than about 1400 ng, less than about 1300 ng, less than about 1200
ng, less than about 1100 ng, less than about 1000 ng, less than
about 900 ng, less than about 800 ng, less than about 700 ng, less
than about 600 ng, less than about 500 ng, less than about 400 ng,
less than about 300 ng, less than about 200 ng, less than about 150
ng, less than about 100 ng, less than about 95 ng, less than about
90 ng, less than about 85 ng, less than about 80 ng, less than
about 75 ng, less than about 70 ng, less than about 65 ng, less
than about 60 ng, less than about 55 ng, less than about 50 ng,
less than about 45 ng, less than about 40 ng, less than about 35
ng, less than about 30 ng, less than about 25 ng, less than about
20 ng, less than about 15 ng, less than about 10 ng, or less than
about 5 ng. In some embodiments, methods provided herein for
selecting a subject for further diagnostic testing include
detecting cfTL (e.g., one or more genetic alterations and/or
aneuploidy) in circulating tumor DNA present in cell-free DNA,
where the circulating tumor DNA represents 100% of the cell-free
DNA. In some embodiments, methods provided herein for selecting a
subject for further diagnostic testing include detecting cfTL
(e.g., one or more genetic alterations and/or aneuploidy) in
circulating tumor DNA present in cell-free DNA, where the
circulating tumor DNA represents less than 100% of the cell-free
DNA, e.g. about 95%, about 90%, about 85%, about 80%, about 75%,
about 70%, about 65%, about 60%, about 55%, about 50%, about 45%,
about 40%, about 35%, about 30%, about 25%, about 20%, about 15%,
about 10%, about 5%, about 4%, about 3%, about 2%, about 1%, about
0.95%, about 0.90%, about 0.85%, about 0.80%, about 0.75%, about
0.70%, about 0.65%, about 0.60%, about 0.55%, about 0.50%, about
0.45%, about 0.40%, about 0.35%, about 0.30%, about 0.25%, about
0.20%, about 0.15%, about 0.10%, about 0.09%, about 0.08%, about
0.07%, about 0.06%, about 0.05% of the cell-free DNA, or less.
[0134] In some embodiments, the diagnostic testing method is a
scan. In some embodiments, the scan is a computed tomography (CT),
a CT angiography (CTA), a esophagram (a Barium swallom), a Barium
enema, a magnetic resonance imaging (MRI), a PET scan, an
ultrasound (e.g., an endobronchial ultrasound, an endoscopic
ultrasound), an X-ray, a DEXA scan.
[0135] In some embodiments, the diagnostic testing method is a
physical examination, such as an anoscopy, a bronchoscopy (e.g., an
autofluorescence bronchoscopy, a white-light bronchoscopy, a
navigational bronchoscopy), a colonoscopy, a digital breast
tomosynthesis, an endoscopic retrograde cholangiopancreatography
(ERCP), an ensophagogastroduodenoscopy, a mammography, a Pap smear,
a pelvic exam, a positron emission tomography and computed
tomography (PET-CT) scan.
[0136] In some embodiments, the diagnostic testing method is a
biopsy (e.g., a bone marrow aspiration, a tissue biopsy). In some
embodiments, the biopsy is performed by fine needle aspiration or
by surgical excision. In some embodiments, the diagnostic testing
methods further includes obtaining a biological sample (e.g., a
tissue sample, a urine sample, a blood sample, a check swab, a
saliva sample, a mucosal sample (e.g., sputum, bronchial
secretion), a nipple aspirate, a secretion or an excretion).
[0137] In some embodiments, the diagnostic testing method includes
determining the presence of a circulating tumor cell. In some
embodiments, the diagnostic testing method includes determining the
complete blood cell count (i.e. the percentage and types of immune
cells). In some embodiments, the diagnostic testing method is a
fecal occult blood test.
[0138] For example, a subject selected for further diagnostic
testing can also be selected for increased monitoring, in which the
subject is administered a diagnostic test at a frequency of twice
daily, daily, bi-weekly, weekly, bi-monthly, monthly, quarterly,
semi-annually, annually, or any at frequency therein. In some
embodiments, a subject selected for further diagnostic testing can
also be selected for increased monitoring, in which the subject is
administered one or more additional diagnostic tests compared to a
subject that has not been selected for further diagnostic testing
and increased monitoring.
Targeted Therapy
[0139] As used herein, the terms "targeted therapy" or "molecularly
targeted therapy" refer to a treatment that recognize and bind to
cell-surface proteins, secreted proteins, peptides, or combinations
thereof, that are associated with cancer cells and/or
cancer-associated cancer cells, without harming non-cancer cells
(e.g., healthy cells). In some embodiments of any of the methods
described herein, the subject has received a targeted therapy.
[0140] In some embodiments, a targeted therapy has a cytotoxic
effect on a cancer cell. In other embodiments, a targeted therapy
has a cytostatic effect on a cancer cell. In some embodiments, a
targeted therapy is an antibody (e.g., a monoclonal antibody, a
humanized chimeric antibody), an antigen-binding fragment thereof,
a small molecule, a small molecule drug conjugate, a small
inhibitory nucleic acid, or a combination thereof.
[0141] In some embodiments, a targeted therapy is a hormone
therapy, a signal transduction inhibitor, a gene expression
modulator, an inducer of apoptosis, an inhibitor of angiogenesis,
or a kinase inhibitor. In some embodiments, the kinase inhibitor is
a tyrosine kinase inhibitor (TKI). In some embodiments, the kinase
inhibitor is a janus kinase inhibitor, an ALK inhibitor, a Bcl-2
inhibitor, a PARP inhibitor, a PI3K inhibitor, a Braf inhibitor, a
MEK inhibitor, or a CDK inhibitor.
[0142] In some embodiments, the targeted therapy is an
anti-angiogenic agent (e.g., bevacizumab (avastin), ramucirumab
(cyramza)).
[0143] In some embodiments, the targeted therapy is an EGFR
inhibitor (e.g., erlotinib (tarceva), afatinib (gilotrif),
gefitinib (iressa), necitumumab (portrazza), cetuximab, osimertinib
(AZD9291, Tagrisso), rociletinib (CO-1686), HM61713 (BI 1482694),
ASP8273, EGF816, PF-06747775). See, e.g., Wang et al. (2016) J.
Hematol Oncol 9:34; Cross et al. (2014) Cancer Discov. 4(9):
1046-61; Walter et al. (2013) Cancer Discov 3(12): 1404-15; Park et
al. (2015) ASCO Meeting Abstract 33(15): 8084; Sequist et al.
(2015) 372(18): 1700-9; Lee et al. (2014) Cancer Res
74(19Supplement): LB-100; Sakagami et al. (2014) Cancer Res 74(19
Supplement): 1728; Goto et al. (2015) ASCO Meeting Abstract
33(15_Suppl):8014; Jia et al. (2016) Cancer Res 76: 1591-602.
[0144] In some embodiments, the targeted therapy is an ALK
inhibitor (e.g., crizotinib (xalkori), ceritinib (zykadia, LDK378),
alectinib (alecensa, RO5424802; CH5424802), brigatinib (alunbrig,
AP26113), lorlatinib (PF-06463922), TSR-011, RXDX-101 (NMS-E628),
X-396, CEP-37440). See, e.g., Tartarone et al. (2017) Med. Oncol.
34(6): 110; Galkin et al. (2007) PNAS 104(1): 270-275; Friboulet et
al. (2014) Cancer Discov. 4(6); 662-73; Chen et al. (2013) 56(14):
5673-5674; Shaw et al. (2014) N. Engl. J. Med. 370(13): 1189-1197;
Sakamoto et al. (2011) Cancer Cell 19(5): 679-690; Squillace et al.
(2013) Cancer Res. 73(8_suppl_: 5655; Mori et al. (2014) 13(2):
329-340; Patnaik et al. (2013) J. Clin. Oncol. 31 (15 suppl); Weiss
et al. (2013) J Thorac Oncol. 8(suppl2): S618; Ardini et al. (2009)
Mol. Cancer Ther. 8(12suppl): A244; Horn et al. (2014) J. Clin.
Oncol. 32(15suppl); Cheng et al. (2012) Mol. Cancer Ther. 11(3):
670-679; Zhang et al. (2011) Cancer Res. 70(8suppl): LB-298; Awad
and Shaw (2014) Clin. Adv. Hematol. Oncol. 12(7): 429-439.
[0145] In some embodiments, the targeted therapy is a heat shock
protein 90 inhibitor (e.g, AUY922, ganetspib, AT13387). See, e.g.,
Pillai et al. (2014) Curr Opin Oncol. 26(2): 159-164; Normant et
al. (2011) Oncogene 30(22): 2581-2586; Sequist et al. (2010) J.
Clin. Oncol. 28(33): 4953-4960; Sang et al. (2013) Cancer Discov.
3(4): 430-443; Felip et al. (2012) Ann Oncol 23(suppl9); Miyajima
et al. (2013) Cancer Res. 73(23): 7022-7033.
[0146] In some embodiments, the targeted therapy is a RET inhibitor
(e.g., cabozantinib (XL184), vandetanib, alectinib, sorafenib,
sunitinib, ponatinib) See, e.g., Drilon et al. (2013) Cancer Discov
3:6305; Gautschi et al. (2013) J. Thorac Oncol 8: e43-4; Kodama et
al. (2014) Mol. Cancer Ther. 13: 2910-8; Lin et al. (2016) J.
Thoracic Oncol. 11(11): 2027-2032; Rosell and Karachaliou (2016)
Lancet 17(12): 1623-1625; Falchook et al. (2016) J. Clin Oncol.
34(15): e141-144; Shaw et al. (2013) Nat Rev Cancer 13: 772-787;
Gozgit et al. (2013) Cancer Res 73 (Suppl. 1): 2084.
[0147] In some embodiments, the targeted therapy is a BRAF
inhibitor (e.g., dabrafenib, vemurafenib). See, e.g., Planchard et
al. (2013) J. Clin. Oncol. 31:8009; Gautschi et al. (2013) Lung
Cancer 82: 365-367; Schmid et al. (2015) Lung Cancer 87: 85-87.
[0148] In some embodiments, the targeted drug therapy is a MET
inhibitor (e.g., onartuzumab, ficlatuzumab, rilotumumab,
tivantinib, crizotinib). See, e.g., Spigel et al. (2014) J. Clin.
Oncol. 32: 8000; Patnail et al. (2014) Br. J. Cancer 111: 272-280;
Gordon et al. (2010): Clin. Cancer Res. 16: 699-710; Sequist et al.
(2011) J. Clin. Oncol. 29: 3307-3315; Zou et al. (2007) Cancer Res.
67: 4408-4417; Ou et al. (2011) J. Thorac. Oncol. 6: 942-946.
[0149] Non-limiting examples of tyrosine kinase inhibitors include:
imatinib, sorafenib, sunitinib, dasatinib, lapatinib, nilotinib,
bortezomib, axitinib, pazopanib, afatinib, crizotinib, erlotinib,
gefitinib, and osimertinib.
[0150] Various other examples of targeted therapies are known in
the art.
Therapeutic Interventions
[0151] In some embodiments, when a targeted therapy is determined
not to be effective in a subject (e.g., using any of the variety of
methods disclosed herein), a therapeutic intervention (e.g., a
therapeutic intervention that is different from the ineffective
targeted therapy) can be administered to the subject. Exemplary
therapeutic interventions include, without limitation, adoptive T
cell therapy (e.g., chimeric antigen receptors and/or T cells
having wild-type or modified T cell receptors), chimeric antigen
receptor (CAR) T cell therapy, radiation therapy, surgery (e.g.,
surgical resection), and administration of one or more
chemotherapeutic agents, administration of immune checkpoint
inhibitors, targeted therapies such as kinase inhibitors (e.g.,
kinase inhibitors that target a particular genetic lesion, such as
a translocation or mutation), signal transduction inhibitors,
bispecific antibodies, and/or monoclonal antibodies. Such
therapeutic interventions can be administered alone or in
combination.
[0152] In some embodiments, the therapeutic intervention can
include an immune checkpoint inhibitor (e.g., a single immune
checkpoint inhibitor or a combination of immune checkpoint
inhibitors). Non-limiting examples of immune checkpoint inhibitors
include nivolumab (Opdivo), pembrolizumab (Keytruda), atezolizumab
(tecentriq), avelumab (bavencio), durvalumab (imfinzi), ipilimumab
(yervoy). See, e.g., Pardoll (2012) Nat. Rev Cancer 12: 252-264;
Sun et al. (2017) Eur Rev Med Pharmacol Sci 21(6): 1198-1205;
Hamanishi et al. (2015) J. Clin. Oncol. 33(34): 4015-22; Brahmer et
al. (2012) N Engl J Med 366(26): 2455-65; Ricciuti et al. (2017) J.
Thorac Oncol. 12(5): e51-e55; Ellis et al. (2017) Clin Lung Cancer
pii: S1525-7304(17)30043-8; Zou and Awad (2017) Ann Oncol 28(4):
685-687; Sorscher (2017) N Engl J Med 376(10: 996-7; Hui et al.
(2017) Ann Oncol 28(4): 874-881; Vansteenkiste et al. (2017) Expert
Opin Biol Ther 17(6): 781-789; Hellmann et al. (2017) Lancet Oncol.
18(1): 31-41; Chen (2017) J. Chin Med Assoc 80(1): 7-14.
[0153] In some embodiments, a therapeutic intervention is adoptive
T cell therapy (e.g., chimeric antigen receptors and/or T cells
having wild-type or modified T cell receptors). See, e.g.,
Rosenberg and Restifo (2015) Science 348(6230): 62-68; Chang and
Chen (2017) Trends Mol Med 23(5): 430-450; Yee and Lizee (2016)
Cancer J. 23(2): 144-148; Chen et al. (2016) Oncoimmunology 6(2):
e1273302; US 2016/0194404; US 2014/0050788; US 2014/0271635; U.S.
Pat. No. 9,233,125; incorporated by reference in their entirety
herein.
[0154] In some embodiments, a therapeutic intervention is a
chemotherapeutic agent. Non-limiting examples of chemotherapeutic
agents include: amsacrine, azacitidine, axathioprine, bevacizumab
(or an antigen-binding fragment thereof), bleomycin, busulfan,
carboplatin, capecitabine, chlorambucil, cisplatin,
cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel,
doxifluridine, doxorubicin, epirubicin, erlotinib hydrochlorides,
etoposide, fiudarabine, floxuridine, fludarabine, fluorouracil,
gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan,
lomustine, mechlorethamine, melphalan, mercaptopurine, methotrxate,
mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,
procarbazine, all-trans retinoic acid, streptozocin, tafluposide,
temozolomide, teniposide, tioguanine, topotecan, uramustine,
valrubicin, vinblastine, vincristine, vindesine, vinorelbine, and
combinations thereof. Additional examples of anti-cancer therapies
are known in the art; see, e.g. the guidelines for therapy from the
American Society of Clinical Oncology (ASCO), European Society for
Medical Oncology (ESMO), or National Comprehensive Cancer Network
(NCCN).
[0155] In some embodiments, the therapeutic intervention can result
in an early onset of remission of a cancer in a subject. In some
embodiments, the therapeutic intervention can result in an increase
in the time of remission of a cancer in a subject. In some
embodiments, the therapeutic intervention can result in an increase
in the time of survival of a subject. In some embodiments, the
therapeutic intervention can result in decreasing the size of a
solid primary tumor in a subject. In some embodiments, the
therapeutic intervention can result in decreasing the volume of a
solid primary tumor in a subject. In some embodiments, the
therapeutic intervention can result in decreasing the size of a
metastasis in a subject. In some embodiments, the therapeutic
intervention can result in decreasing the volume of a metastasis in
a subject. In some embodiments, the therapeutic intervention can
result in decreasing the tumor burden in a subject.
[0156] In some embodiments, the therapeutic intervention can result
in improving the prognosis of a subject. In some embodiments, the
therapeutic intervention can result in decreasing the risk of
developing a metastasis in a subject. In some embodiments, the
therapeutic intervention can result in decreasing the risk of
developing an additional metastasis in a subject. In some
embodiments, the therapeutic intervention can result in decreasing
cancer cell migration in a subject. In some embodiments, the
therapeutic intervention can result in decreasing cancer cell
invasion in a subject. In some embodiments, the therapeutic
intervention can result in a decrease in the time of
hospitalization of a subject. In some embodiments, the therapeutic
intervention can result in a decrease of the presence of cancer
stem cells within a tumor in a subject.
[0157] In some embodiments, the therapeutic intervention can result
in an increase in immune cell infiltration within the tumor
microenvironment in a subject. In some embodiments, the therapeutic
intervention can result in altering the immune cell composition
within the tumor microenvironment of a tumor in a subject. In some
embodiments, the therapeutic intervention can result in modulating
a previously-immunosuppressive tumor microenvironment into an
immunogenic, inflammatory tumor microenvironment. In some
embodiments, the therapeutic intervention can result in a reversal
of the immunosuppressive tumor microenvironment in a subject.
[0158] In some embodiments, the therapeutic intervention can halt
tumor progression in a subject. In some embodiments, the
therapeutic intervention can delay tumor progression in a subject.
In some embodiments, the therapeutic intervention can inhibit tumor
progression in a subject. In some embodiments, the therapeutic
intervention can inhibit immune checkpoint pathways of a tumor in a
subject. In some embodiments, the therapeutic intervention can
immuno-modulate the tumor microenvironment of a tumor in a subject.
In some embodiments, the therapeutic intervention can
immuno-modulate the tumor macroenvironment of a tumor in a
subject.
[0159] In some embodiments of any of the methods described herein,
the subject can be administered a single or multiple doses (e.g.,
two, three, four, five, six, seven, eight, nine, or ten doses) of
any of the therapeutic interventions described herein.
[0160] In some embodiments of any of the methods described herein,
the method can further include administering one or more
therapeutic interventions.
[0161] The term "immunotherapy" refers to a therapeutic treatment
that involves administering to a patient an agent that modulates
the immune system. For example, an immunotherapy can increase the
expression and/or activity of a regulator of the immune system. In
other instances, an immunotherapy can decrease the expression
and/or activity of a regulator of the immune system. In some
instances, an immunotherapy can recruit and/or enhance the activity
of an immune cell. An example of an immunotherapy is a therapeutic
treatment that involves administering at least one, e.g., two or
more, immune checkpoint inhibitors. Exemplary immune checkpoint
inhibitors are CTLA-4 inhibitors, PD-1 inhibitors or PD-L1
inhibitors, or combinations thereof.
[0162] In some instances, the immunotherapy is an immune checkpoint
inhibitor. For example, the immunotherapy can include administering
one or more immune checkpoint inhibitors. In some embodiments, the
immune checkpoint inhibitor is a CTLA-4 inhibitor, a PD-1 inhibitor
or a PD-L1 inhibitor. An exemplary CTLA-4 inhibitor would be, e.g.,
ipilimumab (Yervoy.RTM.) or tremelimumab (CP-675,206). In some
embodiments, the PD-1 inhibitor is pembrolizumab (Keytruda.RTM.) or
nivolumab (Opdivo.RTM.). In some embodiments, the PD-L1 inhibitor
is atezolizumab (Tecentriq.RTM.), avelumab (Bavencio.RTM.) or
durvalumab (Imfinzi.TM.) As used herein, the terms "in combination"
or "combination therapy" describe any concurrent or parallel
treatment with at least two distinct therapeutic agents, e.g.,
administration of any of at least two therapeutic interventions. In
some embodiments of any of the methods described herein, the one or
more therapeutic interventions are administered sequentially or
simultaneously to the subject after the cancer cell has been
detected. For example, the one or more therapeutic interventions
can include chemotherapeutic agents, anti-angiogenic agents,
apoptosis-inducing agents, surgical resection, and radiotherapy. In
some embodiments, combined therapy is an epigenetic therapy (e.g.,
any of the epigenetic therapies described herein) and an
immunotherapy (e.g., any of the immunotherapies described herein).
In some embodiments, the combined therapy is 5-AZA and an immune
checkpoint inhibitor (e.g., anti-PD1 and/or anti-CTLA-4 inhibitor)
(Kim (2014) PNAS 111(32): 11774-1179; Wang (2015) Cancer Immunol.
Res. 3(9): 1030-1041; Juergens et al. (2011) Cancer Discov 1(7):
598-607).
Cancers
[0163] A subject according to any of the methods described herein
can have a cancer that includes, without limitation, lung cancer
(e.g., small cell lung carcinoma or non-small cell lung carcinoma),
papillary thyroid cancer, medullary thyroid cancer, differentiated
thyroid cancer, recurrent thyroid cancer, refractory differentiated
thyroid cancer, lung adenocarcinoma, bronchioles lung cell
carcinoma, multiple endocrine neoplasia type 2A or 2B (MEN2A or
MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia,
breast cancer, colorectal cancer (e.g., metastatic colorectal
cancer), papillary renal cell carcinoma, ganglioneuromatosis of the
gastroenteric mucosa, inflammatory myofibroblastic tumor, or
cervical cancer, acute lymphoblastic leukemia (ALL), acute myeloid
leukemia (AML), cancer in adolescents, adrenal cancer,
adrenocortical carcinoma, anal cancer, appendix cancer,
astrocytoma, atypical teratoid/rhabdoid tumor, basal cell
carcinoma, bile duct cancer, bladder cancer, bone cancer, brain
stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt
lymphoma, carcinoid tumor, unknown primary carcinoma, cardiac
tumors, cervical cancer, childhood cancers, chordoma, chronic
lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML),
chronic myeloproliferative neoplasms, colon cancer, colorectal
cancer, craniopharyngioma, cutaneous T-cell lymphoma, bile duct
cancer, ductal carcinoma in situ, embryonal tumors, endometrial
cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing
sarcoma, extracranial germ cell tumor, extragonadal germ cell
tumor, extrahepatic bile duct cancer, eye cancer, fallopian tube
cancer, fibrous histiocytoma of bone, gallbladder cancer, gastric
cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal
tumors (GIST), germ cell tumor, gestational trophoblastic disease,
glioma, hairy cell tumor, hairy cell leukemia, head and neck
cancer, heart cancer, hepatocellular cancer, histiocytosis,
Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma,
islet cell tumors, pancreatic neuroendocrine tumors, Kaposi
sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal
cancer, leukemia, lip and oral cavity cancer, liver cancer, lung
cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma
of bone, osteocarcinoma, melanoma, Merkel cell carcinoma,
mesothelioma, metastatic squamous neck cancer, midline tract
carcinoma, mouth cancer, multiple endocrine neoplasia syndromes,
multiple myeloma, mycosis fungoides, myelodysplastic syndromes,
myelodysplastic/myeloproliferative neoplasms, myelogenous leukemia,
myeloid leukemia, multiple myeloma, myeloproliferative neoplasms,
nasal cavity and paranasal sinus cancer, nasopharyngeal cancer,
neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer,
oral cancer, oral cavity cancer, lip cancer, oropharyngeal cancer,
osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis,
paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid
cancer, penile cancer, pharyngeal cancer, pheochromosytoma,
pituitary cancer, plasma cell neoplasm, pleuropulmonary blastoma,
pregnancy and breast cancer, primary central nervous system
lymphoma, primary peritoneal cancer, prostate cancer, rectal
cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma,
salivary gland cancer, sarcoma, Sezary syndrome, skin cancer, small
cell lung cancer, small intestine cancer, soft tissue sarcoma,
squamous cell carcinoma, squamous neck cancer, stomach cancer,
T-cell lymphoma, testicular cancer, throat cancer, thymoma and
thymic carcinoma, thyroid cancer, transitional cell cancer of the
renal pelvis and ureter, unknown primary carcinoma, urethral
cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar
cancer, Waldenstrom Macroglobulinemia, and Wilms' tumor.
[0164] In some embodiments of any of the methods described herein,
the subject has non-small cell lung cancer, melanoma, colorectal
cancer, ovarian cancer, or breast cancer.
[0165] In some embodiments of any of the methods described herein,
the subject has the cancer is selected from the group consisting
of: a head and neck cancer, a central nervous system cancer, a lung
cancer, a mesothelioma, an esophageal cancer, a gastric cancer, a
gall bladder cancer, a liver cancer, a pancreatic cancer, a
melanoma, an ovarian cancer, a small intestine cancer, a colorectal
cancer, a breast cancer, a sarcoma, a kidney cancer, a bladder
cancer, a uterine cancer, a cervical cancer, and a prostate cancer.
Various embodiments of such cancers, as therapeutic interventions
appropriate to treat such cancers, are described herein.
Colorectal Cancer
[0166] In some embodiments wherein the subject has colorectal
cancer, the subject may have hereditary colorectal cancer. In some
embodiments, the subject has polyposis (e.g., familial adenomatous
polyposis (FAP) or attenuated FAP (AFAP) (Half et al. (2009)
Orphanet J Rare Dis. 4:22; Knudsen et al. (2003) Fam Cancer
2:43-55). In some embodiments, the subject has a mutation in an
adenomatosis polyposis coli (APC) gene and/or a mutY DNA
glycosylase (MYH) gene (Theodoratou et al. (2010) Br. J. Cancer
103: 1875-1884). In some embodiments, the subject has hereditary
nonpolyposis colorectal cancer (HNPCC; also known as Lynch
Syndrome) (Marra et al. (1995) J. Natl. Cancer Inst 87: 1114-1135).
In some embodiments, the subject has a mutation in a DNA mismatch
repair gene (e.g., mutL homolog 1 (MLHI), mutS homolog 2 (MSH2),
mutS homolog 6 (MSH6) and/or PMS1 homolog 2 (PMS2)). In some
embodiments, the subject has a mutation in an epithelial cell
adhesion molecule (EPCAM) gene. In some embodiments, the subject
has a mutation in an axin-related protein 2 (AXIN2) gene (Lammi et
al. (2004) Am. J. Hum. Genet. 74: 1043-1050). In some embodiments
wherein a colorectal cancer cell has been detected in the subject,
the subject has oligopolyposis, juvenile polyposis syndrome, Cowden
syndromw, Peutz-Jeghers syndrome (Giardiello et al. (2006) Clin.
Gastroenterol. Hepatol. 4:408-415), or serrated polyposis syndrome
(Torlakovic et al. (1996) Gastroenterology 110: 748-755). In some
embodiments, the subject has hereditary mixed polyposis syndrome
(Whitelaw et al. (1997) Gastroenterology 112: 327-334; Tomlinson et
al. (1999) Gastronenterology 116: 789-795).
[0167] In some embodiments wherein the subject has colorectal
cancer, the subject has a colorectal cancer that has at least one
mutation in a gene selected from the group consisting of:
adenomatosis polyposis coli (APC), mutY DNA glycosylase (MYH), mutL
homolog 1 (MLHI), mutS homolog 2 (MSH2), mutS homolog 6 (MSH6),
PMS1 homolog 2 (PMS2), epithelial cell adhesion molecule (EPCAM),
DNA polymerase epsilon (POLE), DNA polymerase delta 1 (POLD1), nth
like DNA glycosylase 1 (NTHL1), bone morphogenetic protein receptor
type 1A (BMPRIA), SMAD family member 4 (SMAD4), phosphatase and
tensin homolog (PTEN), serine/threonine kinase 11 (LKB1, STKI1),
transforming growth factor beta receptor 2 (TGF.beta.RI1),
phosphatidylinositol-4,5-biphosphate-3-kinase catalytic subunit
alpha (PIK3CA), tumor protein p53 (TP53), epidermal growth factor
receptor (EGFR), B-raf proto-oncogene (BRAF),
phosphatidylinositol-4,5-biphosphate-3-kinase (PI3K), A-T rich
interaction domain 1A (ARIDIA), sex determining region Y-bod 9
(SOX9), erb-b2 receptor tyrosine kinase 2 (ERBB2), insulin like
growth factor 2 (IGF2), APC membrane recruitment protein (FAM123B;
AMER1), neuron navigator 2 (NAV2), vacuolar protein sorting 72
homolog (TCFL1; VPS72), N-Ras proto-oncogene (NRAS), and
combinations thereof. See, e.g., Armaghany et al. (2012)
Gastrointest. Cancer Res. 5(1): 19-27; Bulow et al. (2004) Gut 53:
381-386; Zeichner et al. (2012) Clin. Med. Insights Oncol. 6:
315-323; The Cancer Genomic Atlas Network (2012) Nature 487:
330-337; Kemp et al. (2004) Hum. Mol. Genet. 13(suppl_2: R177-R185;
Zouhairi et al. (2011) Gastrointest Cancer Res 4(1): 15-21.
[0168] In some embodiments, the subject has a genetic mutation that
can result in activation of a proto-oncogene (e.g., KRAS). In some
embodiments, the subject has a genetic mutation that can result in
inactivation of a tumor suppressor gene (e.g., 1, 2, 3, 4, 5, 6, at
least 1, at least 2 or at least 3 tumor suppressor genes). In some
embodiments, at least three tumor suppressor genes are inactivated
(e.g., APC, TP53, and loss of heterozygosity of long arm of
chromosome 18). In some embodiments, the subject has a genetic
mutation in a gene involved in the APC/Wnt/.beta.-catenin pathway.
In some embodiments, the genetic mutation is a nonsense mutation or
a frameshift mutation, thereby resulting in a truncated protein. In
some embodiments, the genetic mutation causes microsatellite
instability, epigenetic instability and/or aberrant CpG
methylation.
[0169] In some embodiments of any of the methods described herein
wherein the subject has previously been diagnosed with colorectal
cancer, the subject is administered an additional therapeutic
intervention that specifically targets the genetic modifications
present in the subject's colorectal cancer. In some embodiments,
the subject was previously administered an anti-EGFR monoclonal
antibody (e.g., cetuximab or panitumumab) (Cunningham et al. (2004)
N. Engl. J. Med. 351(4): 337-345). In some embodiments, the
therapeutic invention is an antiangiogenic agent. In some
embodiments, the antiangiogenic agent is bevacizumab (Avastin)
(Hurwitz et al. (2004) N. Engl. J. Med. 350: 2335-2342). In some
embodiments, the antiangiogenic agent is a VEGF inhibitor (e.g.,
aflibercept (Tang et al. (2008) J. Clin. Oncol 26 (May 20 suppl;
abstr 4027); vatalanib (PTK/ZK222584; Hecht et al. (2005) ASCO
Annual Meeting Proceedings J. Clin. Oncol. 23: 16S (abstr. LBA3));
sunitinib (Saltz et al. (2007) J. Clin. Oncol. 25: 4793-4799);
AZD2171 (Rosen et al. (2007) J. Clin. Oncol. 25: 2369-76); AMG 706
(Drevis et al. (2007) 25: 3045-2054)).
[0170] Non-limiting examples of chemotherapy treatments that can be
used in a subject with colorectal cancer include: 5-FU, leucovorin,
oxaliplatin (Eloxatin), capecitabine, celecoxib and sulindac. In
some embodiments, a combination of chemotherapeutic agents is used,
e.g., FOLFOX (5-FU, leucovorin and oxaliplatin), FOLFIRI
(leucovorin, 5-FU and irinotecan (Camptosar), CapeOx (capecitabine
(Xeloda) and oxaliplatin). In some embodiments, the therapeutic
intervention is a mammalian target of rapamycin (mTOR) inhibitor
(e.g., a rapamycin analog (Kesmodel et al. (2007) Gastrointestinal
Cancers Symposium (abstr 234)); RAD-001 (Tabernero et al. (2008) J.
Clin. Oncol. 26: 1603-1610). In some embodiments, the therapeutic
intervention is a protein kinase C antagonist (e.g., enzastaurin
(Camidge et al. (2008) Anticancer Drugs 19:77-84, Resta et al.
(2008) J. Clin. Oncol. 26 (May 20 suppl) (abstr 3529)). In some
embodiments, the therapeutic intervention is an inhibitor of
nonreceptor tyrosine kinase Src (e.g., AZ0530 (Tabernero et al.
(2007) J. Clin. Oncol. 25: 18S (abstr 3520))). In some embodiments,
the therapeutic intervention is an inhibitor of kinesin spindle
protein (KSP) (e.g., ispinesib (SB-715992) (Chu et al. (2004) J.
Clin. Oncol. 22:14S (abstr 2078), Burris et al. (2004) J. Clin.
Oncol. 22: 128 (abstr 2004))).
[0171] In some embodiments, the therapeutic intervention is surgery
(e.g., polypectomy, partial colectomy, colectomy or diverting
colostomy). In some embodiments, adjuvant chemotherapy is further
administered to the subject after surgery (e.g., polypectomy or
partial colectomy). In some embodiments, the therapeutic
intervention is a prophylactic surgery (e.g., colectomy). In some
embodiments, a cancer may be removed by ablation or
embolization.
Ovarian Cancer
[0172] In some embodiments of any of the methods described herein,
the subject may have hereditary ovarian cancer (Petrucelli et al.
(2010) Gen. Med 12:245-259). In some embodiments, the subject has
another genetic condition that may cause ovarian cancer (e.g.,
Lynch syndrome, Peutz-Jeghers syndrome, nevoid basal cell carcinoma
syndrome (NBCCS; also known as Gorlin syndrome), Li-Fraumeni
syndrome or Ataxia-Telangiecstasia (Cancer.Net). In some
embodiments, the subject may have an invasive epithelial ovarian
cancer, an epithelial tumor of low malignant potential (also known
as an atypical proliferating tumor or a borderline tumor), a germ
cell tumor of the ovary (e.g. a malignant germ cell tumor, a
dysgerminoma, an immature teratoma) or a stromal tumor of the
ovary.
[0173] In some embodiments, the subject's ovarian cancer was caused
by a somatic mutation in a gene. In some embodiments, the subject
has a mutation in a gene selected from the group consisting of:
tumor protein p53 (TP53), breast cancer 1 (BRCA1), breast cancer 2
(BRCA2), mutL homolog 1 (MLHI), mutS homolog 2 (MSH2), AKT
serine/threonine kinase 1 (AKTI), BRAC1 associated ring domain 1
(BARD1), BRAC1 interacting protein C-terminal helicase 1 (BRIP1),
epithelial cadherin 1 (CDH1), checkpoint kinase 2 (CHEK2), catenin
beta 1 (CTNNB1), MRE11 homolog (MRE11), mutS homolog 6 (MSH6),
nibrin (NBN), opiod binding protein/cell adhesion molecule like
(OPCML), partner and localizer of BRCA2 (PALB2),
phosphatidylinositol-4,5-biphosphate-3-kinase catalytic subunit
alpha (PIK3CA), PMS1 homolog 2 (PMS2), parkin RBR E3 ubiquitin
protein ligase (PRKN), RAD50 double strand break repair protein
(RAD50), RAD51 recombinase (RAD51), serine/threonine kinase 11
(LKB1, STKI1), neurofibromin (NF1), retinoblastoma 1 (RB1), cyclin
dependent kinase 12 (CDK12), and combinations thereof. See, e.g.,
Kurman et al. (2011) Hum. Pathol. 42(7): 918-31; Nakayama et al.
(2006) Cancer Biol. Ther. 5(7): 779-785; Singer et al. (2003) J.
Natl Cancer Inst 95(6): 484-6; Kuo et al. (2009) Am. J. Pathol.
174(5): 1597-601; Gemignani et al. (2003) Gynecol. Oncol. 90(2):
378-81; Levine et al. (2005) Clin. Cancer Res. 11(8): 2875-8; Wang
et al. (2005) Hum. Mutat. 25(3): 322; Landen et al. (2008) J. Clin.
Oncol. 26(6): 995-1005; Ramus et al. (2015) j Natl Cancer Inst
107(11).
[0174] In some embodiments of any of the methods described herein,
the additional therapeutic intervention is chemotherapy (e.g., any
of the platinum-based chemotherapeutic agents described herein
(e.g., cisplatin, carboplatin), or a taxane (e.g., placitaxel
(Taxol.RTM.) or docetaxel (Taxotere.RTM.). In some embodiments, the
chemotherapeutic agent is an albumin-bound paclitaxel
(nap-paclitaxel, Abraxane.RTM.), altretamine (Hexalen.RTM.),
capecitabine (Xeloda.RTM.), cyclophosphamide (Cytoxan.RTM.),
etoposide (VP-16), gemcitabine (Gemzar.RTM.), ifosfamide
(Ifex.RTM.), irinotecan (CPT-11, Camptosar.RTM.), liposomal
doxorubicin (Doxil.RTM.), melphalan, pemetrexed (Alimta.RTM.),
topotecan, or vinorelbine (Navelbine.RTM.). In some embodiments,
the therapeutic intervention is a combination of chemotherapeutic
agents (e.g., paclitaxel, ifosfamide, and cisplatin; vinblastine,
ifosfamide and cisplatin; etoposide, ifosfamide and cisplatin).
[0175] In some embodiments, the therapeutic intervention is an
epigenetic therapy (see, e.g., Smith et al. (2017) Gynecol. Oncol.
Rep. 20: 81-86). In some embodiments, the epigenetic therapy is a
DNA methyltransferase (DNMT) inhibitor (e.g., 5-azacytidine
(5-AZA), decitabine (5-aza-2'-deoxycytidine) (Fu et al. (2011)
Cancer 117(8): 1661-1669; Falchook et al. (2013) Investig. New
Drugs 31(5): 1192-1200; Matei et al. (2012) Cancer Res. 72(9):
2197-2205). In some embodiments, the DNMT1 inhibitor is NY-ESO-1
(Odunsi et al. (2014) Cancer Immunol. Res. 2(1): 37-49). In some
embodiments, the epigenetic therapy is a histone deacetylase (HDAC)
inhibitor. In some embodiments, the HDAC inhibitor is vorinostat
(Modesitt (2008) 109(2): 182-186) or belinostat (Mackay et al.
(2010) Eur. J. Cancer 46(9): 1573-1579). In some embodiments, the
HDAC inhibitor is given in combination with a chemotherapeutic
agent (e.g., carboplatin (paraplatin), cisplatin, paclitaxel or
docetaxel (taxotere)) (Mendivil (2013) Int. J. Gynecol. Cancer
23(3): 533-539; Dizon (2012) Gynecol. Oncol. 125(2): 367-371; Dizon
(2012) Int J. Gynecol. Cancer 23(3): 533-539).
[0176] In some embodiments, the therapeutic intervention is an
anti-angiogenic agent (e.g., bevacizumab).
[0177] In some embodiments, the therapeutic intervention is a poly
(ADP-ribose) polymerase (PARP)-1 and/or PARP-2 inhibitor. In some
embodiments, the PARP-1 and PARP-2 inhibitor is niraparib (zejula)
(Scott (2017) Drugs doiL10.1007/s40265-017-0752). In some
embodiments, the PARP inhibitor is olaparib (lynparza) or rucaparib
(rubraca).
[0178] In some embodiments, the therapeutic intervention is a
hormone (e.g., a luteinizing-hormone-releasing hormone (LHRH)
agonist). In some embodiments, the LHRH agonist is goserelin
(Zoladex.RTM.) or leuprolide (Lupron.RTM.). In some embodiments,
the therapeutic intervention is an anti-estrogen compound (e.g.,
tamoxifen). In some embodiments, the therapeutic intervention is an
aromatase inhibitor (e.g., letrozole (Femara.RTM.), anastrozole
(Arimidex.RTM.) or exemestane (Aromasin.RTM.).
[0179] In some embodiments, the therapeutic intervention is surgery
(e.g., debulking of the tumor mass, a hysterectomy, a bilateral
salpingo-oophorectomy, an omentectomy). The term "debulking" refers
to surgical removal of almost the entire tumor ("optimally
debulked"). In some embodiments, debulking can include removing a
portion of the bladder, the spleen, the gallbladder, the stomach,
the liver, and/or pancreas. In some embodiments, adjuvant
chemotherapy is further administered to the subject after surgery
(e.g., debulking of the tumor mass, a hysterectomy, a bilateral
salpingo-oophorectomy, an omentectomy). In some embodiments,
adjuvant chemotherapy is administered intra-abdominally
(intraperitoneally). In some embodiments, the therapeutic
intervention is a prophylactic surgery (e.g., a hysterectomy). In
some embodiments, a paracentesis is performed to remove
ascites.
[0180] In some embodiments, the therapeutic intervention is
radiation therapy. In some embodiments, the radiation therapy is
external beam radiation therapy, brachytherapy or a use of
radioactive phosphorus.
Lung Cancer
[0181] In some embodiments of any of the methods described herein,
the subject may have hereditary lung cancer (Gazdar et al. (2014)
J. Thorac. Oncol. 9(4): 456-63). In some embodiments, the subject
has non-small cell-lung cancer (NSCLC) or small cell lung cancer
(SCLC).
[0182] In some embodiments, the subject's lung cancer was caused by
a somatic mutation in a gene. In some embodiments, the subject has
a mutation in a gene selected from the group consisting of: ARIDIA,
AKT anaplastic lymphoma kinase (ALK), BRAF, cyclin dependent kinase
inhibitor 2 (CDKN2A), discoidin domain receptor tyrosine kinase 2
(DDR2), epidermal growth factor receptor (EGFR), fibroblast growth
factor receptor 1 (FGFR1), HER2/ERBB2, kelch like ECH associated
protein 1 (KEAP1) (Singh et al. (2006) PLoS Med 3: e420), KRAS
proto-oncogene (KRAS), MAP kinase/ERK kinase 1 (MEK1), MET
proto-oncogene (MET), MAX gene associated (MGA), myelocytomatosis
oncogene (MYC), NF1, NRAS, neutrophophic receptor tyrosine kinase 1
(NTRK1), PTEN, PIK3CA, RB1, RNA binding motif protein 10 (RBM10),
ret proto-oncogene (RET), Ras like without CAAX 1 (RIT1) (Berger et
al. (2014) Oncogene), Ros proto-oncogene (ROSI), STE domain
containing 2 (SETD2), SWI/SNF related matrix associated actin
dependent regulator of chromatin, subfamily A, member 4 (SMARCA4)
(Medina et al. (2008) Hum. Mutat. 29: 617-622), (SOX2) (Rudin et
al. (2012) Nature Genet. 44: 111-1116), LKB1 (STKI1)
(Sanchez-Cespedees et al. (2002) Cancer Res. 62: 3659-3662), TP53
(Takahashi et al. (1989) Science 246: 491-494), U2 small nuclear
RNA auxillary factor 1 (U2AF1), and combinations thereof. See e.g.,
The Cancer Genome Atlas Research Network (2014) Nature 511:
543-550; Ding et al. (2008) Nature 1069-1075; The Cancer Genome
Atlas Research Network (2012) Nature 489: 519-525; Seo et al.
(2012) Genome Res. 22: 2109-2119; El-Telbany and Ma (2012) Genes
Cancer 3(7-8): 467-480; Marks et al. (2008) Cancer Res. 68:
5524-5528; De Braud et al. (2014) J. Clin. Oncol. 32: 2502;
Rothschild (2015) Cancers 7: 930949.
[0183] In some embodiments, a copy number variation or an oncogenic
chromosomal gene rearrangement (e.g., oncogenic chromosomal
translocation) is detected in a lung cancer cell. Non-limiting
examples of oncogenic chromosomal translocation found in lung
cancer include: EML4-ALK, TFG-ALK, KIF5B-ALK, KLC1-ALK, PTPN3-ALK,
TPR-ALK, HIP1-ALK, STRN-ALK, DCTN1-ALK, SQSTM1-ALK, BIRC6-ALK,
RET-PTC1, KIF4B-RET, CCDC6-RET and NCOA4-RET. See, e.g., Iyevleva
et al. (2015) Cancer Lett. 362(1): 116-121; Wang et al. (2012) J.
Clin Oncol. 30: 4352-9 In some embodiments, the therapeutic
intervention is an anti-angiogenic agent (e.g., bevacizumab
(avastin), ramucirumab (cyramza)).
[0184] In some embodiments, the therapeutic intervention is a
targeted drug therapy. In some embodiments, the targeted drug
therapy is an EGFR inhibitor (e.g., erlotinib (tarceva), afatinib
(gilotrif), gefitinib (iressa), necitumumab (portrazza), cetuximab,
osimertinib (AZD9291, Tagrisso), rociletinib (CO-1686), HM61713 (BI
1482694), ASP8273, EGF816, PF-06747775). See, e.g., Wang et al.
(2016) J. Hematol Oncol 9:34; Cross et al. (2014) Cancer Discov.
4(9): 1046-61; Walter et al. (2013) Cancer Discov 3(12): 1404-15;
Park et al. (2015) ASCO Meeting Abstract 33(15): 8084; Sequist et
al. (2015) 372(18): 1700-9; Lee et al. (2014) Cancer Res
74(19Supplement):LB-100; Sakagami et al. (2014) Cancer Res 74(19
Supplement): 1728; Goto et al. (2015) ASCO Meeting Abstract
33(15_Suppl):8014; Jia et al. (2016) Cancer Res 76: 1591-602.
[0185] In some embodiments, the targeted drug therapy is an ALK
inhibitor (e.g., crizotinib (xalkori), ceritinib (zykadia, LDK378),
alectinib (alecensa, RO5424802; CH5424802), brigatinib (alunbrig,
AP26113), lorlatinib (PF-06463922), TSR-011, RXDX-101 (NMS-E628),
X-396, CEP-37440). See, e.g., Tartarone et al. (2017) Med. Oncol.
34(6): 110; Galkin et al. (2007) PNAS 104(1): 270-275; Friboulet et
al. (2014) Cancer Discov. 4(6); 662-73; Chen et al. (2013) 56(14):
5673-5674; Shaw et al. (2014) N. Engl. J. Med. 370(13): 1189-1197;
Sakamoto et al. (2011) Cancer Cell 19(5): 679-690; Squillace et al.
(2013) Cancer Res. 73(8_suppl_: 5655; Mori et al. (2014) 13(2):
329-340; Patnaik et al. (2013) J. Clin. Oncol. 31 (15 suppl); Weiss
et al. (2013) J Thorac Oncol. 8(suppl2): S618; Ardini et al. (2009)
Mol. Cancer Ther. 8(12suppl): A244; Horn et al. (2014) J. Clin.
Oncol. 32(15suppl); Cheng et al. (2012) Mol. Cancer Ther. 11(3):
670-679; Zhang et al. (2011) Cancer Res. 70(8suppl): LB-298; Awad
and Shaw (2014) Clin. Adv. Hematol. Oncol. 12(7): 429-439.
[0186] In some embodiments, the targeted drug therapy is a heat
shock protein 90 inhibitor (e.g, AUY922, ganetspib, AT13387). See,
e.g., Pillai et al. (2014) Curr Opin Oncol. 26(2): 159-164; Normant
et al. (2011) Oncogene 30(22): 2581-2586; Sequist et al. (2010) J.
Clin. Oncol. 28(33): 4953-4960; Sang et al. (2013) Cancer Discov.
3(4): 430-443; Felip et al. (2012) Ann Oncol 23(suppl9); Miyajima
et al. (2013) Cancer Res. 73(23): 7022-7033.
[0187] In some embodiments, the targeted drug therapy is a RET
inhibitor (e.g., cabozantinib (XL184), vandetanib, alectinib,
sorafenib, sunitinib, ponatinib) See, e.g., Drilon et al. (2013)
Cancer Discov 3:6305; Gautschi et al. (2013) J. Thorac Oncol 8:
e43-4; Kodama et al. (2014) Mol. Cancer Ther. 13: 2910-8; Lin et
al. (2016) J. Thoracic Oncol. 11(11): 2027-2032; Rosell and
Karachaliou (2016) Lancet 17(12): 1623-1625; Falchook et al. (2016)
J. Clin Oncol. 34(15): e141-144; Shaw et al. (2013) Nat Rev Cancer
13: 772-787; Gozgit et al. (2013) Cancer Res 73 (Suppl. 1):
2084.
[0188] In some embodiments, the targeted drug therapy is a BRAF
inhibitor (e.g., dabrafenib, vemurafenib). See, e.g., Planchard et
al. (2013) J. Clin. Oncol. 31:8009; Gautschi et al. (2013) Lung
Cancer 82: 365-367; Schmid et al. (2015) Lung Cancer 87: 85-87.
[0189] In some embodiments, the targeted drug therapy is a MET
inhibitor (e.g., onartuzumab, ficlatuzumab, rilotumumab,
tivantinib, crizotinib). See, e.g., Spigel et al. (2014) J. Clin.
Oncol. 32: 8000; Patnail et al. (2014) Br. J. Cancer 111: 272-280;
Gordon et al. (2010): Clin. Cancer Res. 16: 699-710; Sequist et al.
(2011) J. Clin. Oncol. 29: 3307-3315; Zou et al. (2007) Cancer Res.
67: 4408-4417; Ou et al. (2011) J. Thorac. Oncol. 6: 942-946.
[0190] In some embodiments, the therapeutic intervention is
administration of an immunotherapy. See, e.g., Smasundaram and
Burns (2017) J. Hematol. Oncol. 10:87. In some embodiments, the
immunotherapy is an anti-PD-1 agent (e.g., nivolumab) (Brahmer et
al. (2012) N. Engl. J. Med. 366(26): 2455-2465; Gettinger et al.
(2016) J. Clin. Oncol. 34(25)), pembrolizumab (Keytruda) (Garon et
al. (2015) N. Engl. J. Med. 372(21): 2018-2028), durvalumab),
nivolumab (opdivo)). In some embodiments, the immunotherapy is an
anti-PD-L1 agent (e.g., atezolizumab (Fehrenbacher et al. (2016)
Lancet 387(10030): 1837-1846, Rittmeyer et al. (2017) Lancet
389(10066): 255-265); atezolizumab (Tecentriq)). In some
embodiments, the immunotherapy is an anti-CTLA-4 agent (e.g.,
ipilimumab or tremlimumab). In some embodiments, the immunotherapy
is a combination therapy of an anti-PD-1 agent and an anti-CTLA-4
agent (e.g., nivolumab and ipilimumab (Herbset et al. (2015) 21(7):
1514-1524), pembrolizumab and ipilimumab (Gubens et al. (2016) ASCO
Meeting Abstracts 34(15_suppl):9027), durvalumab and tremlimumab
(NCT02542293. Study of 1st line therapy study of MEDI4736 with
tremelimumab versus SoC in non-small-cell lung cancer (NSCLC)
(NEPTUNE)).
[0191] In some embodiments, the immunotherapy is given in
combination with a chemotherapeutic agent (e.g., Rizvi et al.
(2016) J. Clin. Oncol. 34(25): 2969-79; Hall et al. (2016) ASCO
Meeting Abstracts. 34(15_suppl):TPS9104).
[0192] In some embodiments, the therapeutic intervention is
chemotherapy (e.g., cisplatin, carboplatin, paclitaxel,
albumin-bound paclitaxel, docetaxel, gemcitabine, vinorelbine,
irinotecan, etoposide, vinblastine or pemetrexed (alimta)). In some
embodiments, the therapeutic intervention is a combination of at
least two chemotherapeutic agents.
[0193] In some embodiments, the therapeutic intervention is surgery
(e.g., a wedge resection (i.e. removal of a small section of
diseased lung and a margin of healthy tissue); a segmental
resection (segmentectomy) (i.e. removal of a larger portion of
lung, but not an entire lobe); a lobectomy (i.e. removal of an
entire lobe of one lung); a pneumonectomy (i.e. removal of an
entire lung)), or a sleeve resection. The extent of surgical
removal will depend on the stage of lung cancer and overall
prognosis. In some embodiments, surgery is carried out by
video-assisted thoracic surgery (VATS). In some embodiments, the
therapeutic intervention is radiofrequency ablation (RFA).
[0194] In some embodiments, the therapeutic intervention is
radiation therapy. In some embodiments, the radiation therapy is
external beam radiation therapy (e.g., three-dimensional conformal
radiation therapy (3D-CRT), intensity modulated radiation therapy
(IMRT), stereotactic body radiation therapy (SBRT), brachytherapy
or a use of radioactive phosphorus.
[0195] In some embodiments, the therapeutic intervention further
comprises palliative care. In some embodiments, palliative care
includes removal of pleural effusion by thoracentesis, pleurodesis
or catheter placement. In some embodiments, palliative care
includes removal of pericardial effusion by pericardiocentesis, a
pericardial window. In some embodiments, the therapeutic
intervention is photodynamic therapy (PDT), laser therapy or stent
placement.
Breast Cancer
[0196] In some embodiments of any of the methods described herein,
the subject may have hereditary breast cancer (Peters et al. (2017)
Gynecol Oncol pii: S0090-8258(17)30794-1). In some embodiments, the
subject may have triple negative breast cancer (estrogen receptor
negative, progesterone receptor negative and HE2-negative), hormone
receptor positive (estrogen and/or progesterone receptor positive)
breast cancer, hormone receptor negative (estrogen and/or
progesterone receptor negative) breast cancer, HER2 positive breast
cancer, HER2 negative breast cancer, inflammatory breast cancer or
metastatic breast cancer.
[0197] In some embodiments wherein a breast cancer cell has been
detected in the subject, the subject has at least one mutation in a
gene selected from the group consisting of: BRCA1, BRCA2, ATM,
CHD1, CHEK2, PALB2, STKI1, TP53, HER2 (ERBB2), CDK416, AKTI, GATA
binding protein 3 (GATA3), RB1, lysine methyltransferase 2C (MLL3),
mitogen-activated protein kinase 1 (MAP3K1), CDKN1B, T-box3(TBX3),
runt related transcription factor 1 (RUNX1), core binding factor
beta (CBFB), phosphoinositide-3-kinase regulatory subunit 1
(PIK3R1), protein tyrosine phosphatase non-receptor type 22
(PTPN22), protein tyrosine phosphatase receptor type D (PTPRD),
NF1, splicing factor 3b subunit 1 (SF3B1), cyclin D3 (CCND3), T-box
5 (TBX5), CCCTC-binding factor (CTCF), forkhead box A1 (FOX1),
PI3KCA, PTEN, mitogen-activated protein kinase 4 (MAP2K4), and
combinations thereof. See, e.g., Nik-Zainal et al. (2016) Nature
534: 47-54; Bergamaschi et al. (2008) J. Pathol. 214: 357-367;
Pleasance et al. (2010) Nature 463: 191-196; The Cancer Genome
Atlas Network (2012) Nature 490:61-70; Usary et al. (2004) Oncogene
23: 7669-7678; Bachman et al. (2004) Cancer Biol. Ther. 3: 772-775;
Saal et al. (2008) Nature Genet 40: 102-107; Troester et al. (2006)
BMC Cancer 6: 276; Chandriani et al. (2009) PLoS One 4: e6693;
Matsuda et al. (2017) Breast Cancer Res Treat 163(2): 263-272.
[0198] In some embodiments, the targeted drug therapy is a HER2
inhibitor (e.g., trastuzumab (Herceptin), pertuzumab (perjeta);
ado-trastuzumab emtansine (T-DM1; Kadcyla); lapatinib (Tykerb),
neratinib). See, e.g., Baselga et al. (2012) N Engl J Med 366:
109-119; Konecny et al. (2006) Cancer Res 66: 1630-1639, Xia et al.
(2007) Cancer Res. 67: 1170-1175; Gomez et al. (2008) J Clin Oncol
26: 2999-30005; Wong et al. (2009) Clin. Cancer Res. 15: 2552-2558;
Agus et al. (2002) Cancer Cell 2: 127-137; Lewis Philips et al.
(2008) Cancer Res 68: 9280-9290.
[0199] In some embodiments, the targeted drug therapy is a
cyclin-dependent kinase inhibitor (e.g., a CDK4/6 inhibitor (e.g.,
palbociclib (Ibrance.RTM.), ribociclin (Kisqali.RTM.), abemaciclib)
(Turner et al. (2015) N Engl J Med 373: 209-219; Finn et al. (2016)
N Eng J Med 375: 1925-1936; Ehab and Elbaz (2016) Breast Cancer 8:
83-91; Xu et al. (2017) J Hematol. Oncol. 10(1): 97; Corona et al.
(2017) Cri Rev Oncol Hematol 112: 208-214; Barroso-Sousa et al.
(2016) Breast Care 11(3): 167-173)).
[0200] In some embodiments, the targeted drug therapy is a PARP
inhibitor (e.g., olaparib (AZD2281), veliparib (ABT-888), niraparib
(MK-4827), talazoparib (BMN-673), rucaparib (AG-14699), CEP-9722)
See, e.g., Audeh et al. (2010) Lancet 376: 245-251; Fong et al.
(2009) N Engl J Med 361: 123-134; Livrahi and Garber (2015) BMC
Medicine 13: 188; Kaufamn et al. (2015) J Clin. Oncol. 33: 244-250;
Gelmon et al. (2011) Lancet Oncol. 12: 852-61; Isakoff et al.
(2011) Cancer Res 71:P3-16-05; Sandhu et al. (2013) Lancet Oncol
14:882-92; Tutt et al. (2010) Lancet 376: 235-44; Somlo et al.
(2013) J. Clin. Oncol. 31: 1024; Shen et al. (2013) CLin. Cancer
Res. 19(18): 5003-15; Awada et al. (2016) Anticancer Drugs 27(4):
342-8.
[0201] In some embodiments, the targeted drug therapy is a mTOR
inhibitor (e.g., everolimus (afinitor)). See, e.g., Gong et al.
(2017) Oncotarget doi: 10.18632/oncotarget.16336; Louseberg et al.
(2017) Breast Cancer 10: 239-252; Hare and Harvey (2017) Am J
Cancer Res 7(3): 383-404.
[0202] In some embodiments, the targeted drug therapy is a heat
shock protein 90 inhibitor (e.g., tanespimycin) (Modi et al. (2008)
J. Clin Oncol. 26: s1027; Miller et al. (2007) J. Clin. Oncol.
25:s1115; Schulz et al. (2012) J Exp Med 209(2): 275-89).
[0203] In some embodiments, the targeted drug therapy further
includes a bone-modifying drug (e.g., a bisphosphonate or denosumab
(Xgeva)). See, e.g., Ethier et al. (2017) Curr Oncol Rep 19(3): 15;
Abdel-Rahman (2016) Expert Rev Anticancer Ther 16(8): 885-91.
[0204] In some embodiments, the therapeutic intervention is a
hormone (e.g., a luteinizing-hormone-releasing hormone (LHRH)
agonist). In some embodiments, the LHRH agonist is goserelin
(Zoladex.RTM.) or leuprolide (Lupron.RTM.). In some embodiments,
the therapeutic intervention is an anti-estrogen compound (e.g.,
tamoxifen, fulvestrant (faslodex)). In some embodiments, the
therapeutic intervention is an aromatase inhibitor (e.g., letrozole
(Femara.RTM.), anastrozole (Arimidex.RTM.) or exemestane
(Aromasin.RTM.).
[0205] In some embodiments, the therapeutic intervention is surgery
(e.g., a lumpectomy, a single mastectomy, a double mastectomy, a
total mastectomy, a modified radical mastectomy, a sentinel lymph
node biopsy, an axillary lymph node dissection; breast-conserving
surgery). The extent of surgical removal will depend on the stage
of breast cancer and overall prognosis.
[0206] In some embodiments, the therapeutic intervention is
radiation therapy. In some embodiments, the radiation therapy is
partial breast irradiation or intensity-modulated radiation
therapy.
[0207] In some embodiments, the therapeutic intervention is
chemotherapy (e.g., capecitabine (xeloda), carboplatin
(paraplatin), cisplatin (platinol), cyclophosphamide (neosar),
docetaxel (docefrez, taxotere), doxorubicin (Adriamycin), pegylated
liposomal doxorubicin (doxil), epirubicin (ellence), fluorouracil
(5-FU, adrucil), gemcitabine (gemzar), methotrexate, paclitaxel
(taxol), protein-bound paclitaxel (abraxane), vinorelbine
(navelbine), eribulin (halaven), or ixabepilone (ixempra)). In some
embodiments, the therapeutic intervention is a combination of at
least two chemotherapeutic agents (e.g., doxorubicin and
cyclophosphamide (AC); epirubicin and cyclophosphamide (EC);
cyclophosphamide, doxorubicin and 5-FU (CAF); cyclophosphamide,
epirubicin and 5-FU (CEF); cyclophosphamide, methotrexate and 5-FU
(CMF); epirubicin and cyclophosphamide (EC); docetaxel, doxorubicin
and cyclophosphamide (TAC); docetaxel and cyclophosphamide
(TC).
[0208] Non-limiting aspects of these methods are described below,
and can be used in any combination without limitation. Additional
aspects of these methods are known in the art.
EXAMPLES
[0209] The invention is further described in the following
examples, which do not limit the scope of the claims.
Example 1. Dynamics of Cell-Free Tumor Load
[0210] Serial blood was drawn from fifteen advanced non-small cell
lung cancer (NSCLC) patients undergoing treatment with targeted
tyrosine kinase inhibitors (TKIs), afatinib or osimertinib, to
directly detect somatic sequence and structural alterations in
cfDNA, to monitor ctDNA dynamics during therapy, to determine
cell-free tumor burden, and to predict clinical outcome (FIG. 1).
Liquid biopsies were obtained from 11 radiographic responders and
four radiographic non-responders immediately prior to treatment
(baseline), 6-22 days post treatment, and at serial time points
until disease progression. The ultrasensitive targeted error
correction sequencing (TEC-Seq) approach (6) was used as well as
whole genome sequencing to identify tumor-derived sequence
alterations and chromosomal copy number changes in cell-free DNA
(cfDNA). The dynamics of alterations were identified, and developed
a non-invasive measure of cfTL to evaluate real-time response to
treatment. Next, changes in cfTL were evaluated within hours to
days after treatment compared to baseline and assessed whether cfTL
could serve as a marker of patient outcome.
[0211] For each patient, .about.5 ml of plasma were collected
immediately prior to therapy (baseline), at 6-22 days after therapy
initiation (median 16 days), and at additional serial time points
until disease progression was confirmed by radiographic assessment
(Tables S1 and S2). Target lesions were evaluated before therapy
with CT/MRI imaging and approximately every six weeks until disease
progression. Based on response assessment by RECIST 1.1 of the
initial surveillance scans after therapy, nine of fifteen patients
experienced partial response, one had stable disease, four
progressed, and one patient had non-measurable disease due to
bone-only metastases (Table S1). The patient with stable disease at
the initial scan achieved a partial response on later scans.
[0212] To analyze changes in cfDNA in these patients and capture
the clonal heterogeneity of metastatic disease, we developed a
combined comprehensive approach for analysis of both sequence and
chromosomal changes. For sequence analyses, the targeted error
correction sequencing (TEC-Seq) approach was used to evaluate 58
well-known cancer driver genes (FIG. 1 and Tables S3, S4 and S5)
(6). This method is based on targeted capture and deep sequencing
(>30,000.times.) of DNA fragments to provide a high degree of
specificity across 80,930 bp of coding gene regions and enables
identification of tumor-specific alterations in ctDNA while
distinguishing these from amplification and sequencing artifacts,
germline changes, or alterations related to blood cell
proliferation that may be present in cfDNA (6).
[0213] To evaluate chromosomal changes that may be present in
ctDNA, whole genome sequences were used obtained from off-target
fragments that were not captured during analysis of targeted
regions in manner similar to other genome wide copy number
analyses, including Digital Karyotyping and related NGS approaches
(10, 26, 27). The most aberrant alterations in the genome
representation of individual chromosome arms were used to construct
a plasma aneuploidy score (PA-Score) that was evaluated to detect
changes in ctDNA during therapy (Table S6).
[0214] ctDNA was evaluated in all patients at baseline
(pre-treatment) and 6-22 days after the initiation of therapy. In
the blood draws that were analyzed, sequence alterations were
detected in 14 of 15 cases. At the baseline time point, patients
had an average of 3.6 tumor-specific somatic mutations, affecting
14 driver genes, ranging from one to 14 alterations per case (Table
S5). At least one targetable mutation in either EGFR or ERBB2 was
detected in each case analyzed, with ctDNA mutant allele fractions
ranging from 0.10% to 53.71% (Table S5). Eight out of the 11
patients treated with osimertinib had EGFR T790M acquired
resistance mutations in the circulation at baseline, with ctDNA
mutant allele fractions ranging from 0.17% to 10.09% (Table S5),
consistent with their previous treatment with EGFR TKIs (Table S1).
Previously described alterations in genes involved in blood cell
proliferation (28-32) were observed in nine patients across all
time points analyzed, and were removed from further analyses (Table
S7). Chromosomal abnormalities were detected in 13 of 15 cases. In
most patients, multiple chromosomal arms were aberrant (FIG. 2,
FIGS. 6A-M, Table S6), resulting in PA-Scores ranging from 1.3 to
14.9 at the baseline blood sample. Through the combined analyses,
either a tumor-derived sequence or chromosomal change or both types
of alterations were detected in all 15 cases.
[0215] Based on the alterations observed in cfDNA through analyses
of multiple genes, a new metric, termed cell-free tumor load
(cfTL), was developed, which is defined as the contribution of the
most abundant alterations in cfDNA at any particular time point
during the course of tumor evolution (FIG. 1 and Tables S5 and S6).
In this study, the most abundant alterations were typically in
driver genes targeted by the TKIs utilized (e.g., EGFR and ERBB2).
A tiered approach to evaluate ctDNA levels was used, first using
cfTL levels based on sequence changes, and then PA-Scores based on
chromosomal changes if sequence alterations were not present. This
approach had the benefit of providing a comprehensive assessment of
tumor-derived alterations that would represent overall tumor burden
during the course of disease and selective pressure of therapeutic
interventions.
[0216] All patients with an initial objective radiographic response
to targeted therapy displayed a rapid and dramatic reduction of
cfTL (>95% decrease, P<0.01, Wilcoxon signed rank test) at
6-22 days after initiation of therapy (FIG. 2, FIGS. 6A-M and Table
S). FIG. 2 depicts a representative patient with metastatic disease
(CGPLLU2) who had a rapid decline of cfTL from baseline to day 10.
This patient exhibited a progression-free survival of 7.0 months on
osimertinib therapy, then subsequently developed resistance in the
primary lung lesion. In patients with radiographic response or
stable disease, mutant allele concentrations were reduced from an
average of 10.80% at baseline to 0.20% at 6-22 days after treatment
(>95% reduction) (FIG. 3A and Table S5) (P=0.002, Wilcoxon
signed rank test). Likewise, PA-Scores decreased in responders
(average decrease of 92%, P=0.002, Wilcoxon signed rank test),
including in patient CGPLLU97 who had no sequence alterations
detected in the plasma (FIG. 3B).
[0217] In contrast, all four patients who were radiographic
non-responders to targeted therapy experienced limited variation in
cfTL, as measured through both sequence and chromosomal
alterations, ranging from an average mutant allele fraction of
6.38% at baseline to 5.70% at 6-22 days after treatment (FIGS. 2,
3A, 3B and FIGS. 6A-M) (P=0.6, Wilcoxon signed rank test).
[0218] In addition to changes in cfTL, the average number of
observed alterations also decreased in responders from 4.0 to 1.2
mutations per patient (P=0.006, Wilcoxon signed rank test), while
non-responders had no change in the number of mutations observed
during therapy (2.5 mutations per patient both before and after
therapy) (FIG. 3C).
[0219] Clinical NGS testing performed to identify alterations in
tumor tissue or plasma during the care of these patients
independently confirmed 86% of the changes detected in this study
(FIG. 7). These observations suggested that both ctDNA levels and
clonal heterogeneity were dramatically reduced at early time points
in responding patients due to therapeutic selective pressure. In
non-responding patients, the emergence and growth of tumor
subclones could be detected earlier than radiographic
progression.
Example 2. Analysis of cfDNA within Hours of Therapy
[0220] For a subset of patients, multiple follow-up blood draws
were evaluated at extremely early time points in therapy. An
immediate time point within the same day at 4-12 hours after the
initiation of the first dose of treatment was available for four
patients who experienced a partial radiographic response on the
first or second scan (CGPLLU2, CGPLLU14, CGPLLU86, and CGPLLU99),
one clinical responder classified with non-measurable disease by
RECIST 1.1 (CGLU315), and one patient with progressive disease
(CGLU294). In five of the six patients for whom immediate time
points were evaluated, increasing ctDNA levels allowed for the
identification of seven tumor-derived alterations not previously
detected at baseline including the targetable EGFR 746ELREATS>D
clone in patient CGPLLU86 detected at a mutant allele fraction of
0.19% (FIG. 4A). Mutant allele fractions of the newly detected
clones ranged from 0.16% to 1.70% with an average of 0.63% and
suggested that these alterations were likely below the limit of
detection at baseline and were detected due to an increase in ctDNA
levels. Remarkably, five of the six patients had newly emerging
alterations within the same day after initiation of therapy
compared to emerging alterations in two patients detected at
earlier time points within 188 days prior to therapy. Evaluating
the relative rate of emerging mutations in a Bayesian statistical
model, we estimated a 110-fold increase in the rate of emerging
mutations comparing post-treatment to pre-treatment levels (99% CI:
13-732). Overall cfDNA amounts remained relatively constant between
baseline and time points 4-12 hours after treatment indicating that
changes in ctDNA levels occurred due to changes in the relative
abundance of mutated clones within cfDNA (FIG. 4B). These
observations suggested that the emergence of novel ctDNA variants
were related to early effects of therapy and are consistent with
studies showing BIM-mediated apoptosis in responsive tumors 6-48
hours after exposure to EGFR inhibitors (33, 34).
Example 3. Cell-Free Tumor Load and Clinical Outcome
[0221] The dynamic cfTL changes observed at early time points after
treatment initiation were associated with differences in clinical
outcome. cfTL levels at day 6-22 were bimodal, with the lower group
clustering at an average reduction in cfTL of 99.8% and the higher
group having an average increase in cfTL of 0.8% (FIG. 5A). ctDNA
responders were defined as those with reduction in cfTL levels
within three standard deviations of average reduction of the lower
group (greater than 98.7%) while non-responders were below this
threshold. Three of eight patients who developed a complete ctDNA
response (cfTL reduction of 100%) at day 6-22 experienced
progression-free survival longer than one year, while two continue
to respond, but have not yet reached one year of follow-up (FIG. 5B
and FIG. 8).
[0222] Three patients had different measures of response between
ctDNA analyses at day 6-22 and initial scans after therapy (FIG.
5B, FIG. 9 and FIG. 10). For example, one of the ctDNA responders
(CGPLLU86) with a cfTL reduction of 100% at day 7 and 17 post
treatment who had a progression-free survival of 12.4 months was
classified by RECIST as having stable disease at 38 days post
treatment and achieved a maximal reduction of 46% only by the ninth
CT scan (371 days) (FIG. 5B, FIG. 8 and FIG. 9). Similarly, another
complete ctDNA responder (CGLU315) assessed at day 20 post
treatment was classified as radiographically non-measurable yet
continues to have a favorable clinical response and remains on
treatment as of last follow-up (FIG. 5B FIG. 8). Although patient
CGPLLU18 was classified by RECIST as a partial responder, cfTL
analyses revealed that the patient was a ctDNA non-responder and
had a limited therapeutic benefit (progression-free survival of 3.9
months) (FIG. 5B, FIG. 8 and FIG. 9).
[0223] Overall, we observed a significantly longer median
progression-free survival for ctDNA responders at day 6-22 compared
to ctDNA non-responders (10.8 months vs. 2.0 months, P<0.001,
FIG. 5C). Importantly, cfTL reduction at day 6-22 was as accurate
of a predictor of outcome as initial CT imaging performed an
average of 48 days after initiation of therapy (FIG. 5C, FIG. 5D
and FIG. 11.
[0224] Faster and predictive determinants of patient response can
aid in navigating adaptive therapeutic strategies to reduce
toxicity, identify early resistance to targeted therapy, and enable
the consideration of combinatorial approaches early in a patient's
therapy. Additionally, as occurred in patient CGPLLU86, these data
indicate the utility of a single dose of treatment to elicit levels
of ctDNA high enough to identify possible tumor-derived
alterations.
Example 4. Methods
Patient and Sample Characteristics
[0225] Fifteen patients with metastatic non-small cell lung cancer
undergoing treatment with tyrosine kinase inhibitors (TKIs) were
included in our study. Clinical and pathological characteristics
for all patients are summarized in Tables S1 and S2, and tumor load
dynamics are shown in FIG. 2 and in FIGS. 6A-M. Patient enrollment
and genomic studies were conducted in accordance with the
Declaration of Helsinki, were approved by the Institutional Review
Board (IRB) and patients provided written informed consent for
sample acquisition for research purposes.
[0226] Of the fifteen patients, four were initially diagnosed with
stage IIA or IIIA disease and treated with chemotherapy followed by
osimertinib upon disease progression, and eleven were initially
diagnosed with advanced disease and either treated with
chemotherapy prior to osimertinib (n=2) or treated directly with
first-line TKI therapy (n=9). Of the nine patients who received
first-line TKI treatment, five received osimertinib and four
received afatinib. Osimertinib was dosed at 80 mg PO daily and
afatinib at 40 mg PO daily (Table S).
[0227] The response evaluation criteria in solid tumors (RECIST)
version 1.1 (35) were used for assessment of response. Of the
fifteen patients analyzed, nine achieved a partial response based
on their initial CT assessment post treatment while one patient
exhibited stable disease and one patient had unmeasurable disease.
Upon follow-up of these eleven patients, eight eventually
experienced disease progression while two continue to derive
clinical benefit from targeted inhibition, one having continued
response and one having unmeasurable disease. Four of fifteen
patients had progressive disease evident on the initial CT
assessment post treatment and did not exhibit radiographic response
by RECIST 1.1 (Table S1).
[0228] For all patients, serial blood draws were collected over the
course of treatment with targeted inhibition for isolation of
plasma and extraction of cell-free DNA (cfDNA) for genomic
analyses. Time points were analyzed immediately prior to treatment
for baseline assessment as well as 6-22 days post treatment and at
serial time points until disease progression (Table S2).
Sample Preparation and Next-Generation Sequencing of cfDNA
[0229] Whole blood was collected in K2 EDTA tubes or Streck tubes
and processed immediately or within 2 hours after storage at
4.degree. C. for EDTA tubes or room temperature for Streck tubes,
respectively. Plasma and cellular components were separated by
centrifugation at 800 g for 10 minutes at 4.degree. C. Plasma was
centrifuged a second time at 18,000 g at room temperature to remove
any remaining cellular debris and stored at -80.degree. C. until
the time of DNA extraction. DNA was isolated from plasma using the
QiAmp.RTM. Circulating Nucleic Acids Kit (Qiagen GmbH) and eluted
in LoBind tubes (Eppendorf.RTM.AG). Concentration and quality of
cfDNA was assessed using the Bioanalyzer 2100 (Agilent
Technologies).
[0230] TEC-Seq next-generation sequencing cell-free DNA libraries
were prepared from 11 to 350 ng of cfDNA. Genomic libraries were
prepared as previously described (6). Briefly, the NEBNext.RTM. DNA
Library Prep Kit for Illumina (New England Biolabs (NEB)) was used
with four main modifications to the manufacturer's guidelines: i)
The library purification steps utilized the on-bead Agencourt.RTM.
Ampure.RTM. XP approach, ii) reagent volumes were adjusted
accordingly to accommodate the on-bead strategy, iii) a pool of 8
unique Illumina dual index adapters with 8 bp barcodes were used in
the ligation reaction, and iv) cfDNA libraries were amplified with
Hotstart Phusion.TM. Polymerase. Genomic library preparation was
performed as previously described (6). Concentration and quality of
cfDNA genomic libraries were assessed using the Bioanalyzer 2100
(Agilent Technologies).
[0231] Targeted capture was performed using the Agilent SureSelect
reagents and a custom set of hybridization probes targeting 58
genes (Table 1 and Table S3) per the manufacturer's guidelines. The
captured library was amplified with HotStart Phusion.TM. Polymerase
(NEB). The concentration and quality of captured cfDNA libraries
was assessed on the Bioanalyzer (Agilent Technologies). TEC-seq
libraries were sequenced using 100-bp paired end runs on the
Illumina HiSeq 2500 (Illumina).
TABLE-US-00001 TABLE 1 Genes Analyzed Gene Region Analyzed ABL1
Specific Exons AKT1 Specific Exons ALK Full Coding Region APC
Specific Exons AR Full Coding Region ATM Specific Exons BRAF Full
Coding Region CDH1 Specific Exons CDK4 Full Coding Region CDK6 Full
Coding Region CDKN2A Specific Exons CSF1R Specific Exons CTNNB1
Specific Exons DNMT3A Specific Exons EGFR Full Coding Region ERBB2
Specific Exons ERBB4 Full Coding Region ESR1 Full Coding Region
EZH2 Specific Exons FBXW7 Specific Exons FGFR1 Specific Exons FGFR2
Specific Exons FGFR3 Specific Exons FLT3 Specific Exons GNA11
Specific Exons GNAQ Specific Exons GNAS Specific Exons HNF1A
Specific Exons HRAS Full Coding Region IDH1 Specific Exons IDH2
Specific Exons JAK2 Full Coding Region JAK3 Specific Exons KDR
Specific Exons KIT Full Coding Region KRAS Full Coding Region
MAP2K1 Specific Exons MET Specific Exons MLH1 Specific Exons MPL
Specific Exons MYC Specific Exons NPM1 Specific Exons NRAS Full
Coding Region PDGFRA Full Coding Region PIK3CA Full Coding Region
PIK3R1 Specific Exons PTEN Full Coding Region PTPN11 Specific Exons
RB1 Specific Exons RET Specific Exons SMAD4 Specific Exons SMARCB1
Specific Exons SMO Specific Exons SRC Specific Exons STK11 Full
Coding Region TERT Specific Exons TP53 Full Coding Region
Primary Processing of Next-Generation Sequencing Data and
Identification of Putative Somatic Mutations
[0232] Primary processing of next-generation sequence data for
analyses of sequence alterations in cfDNA samples was performed as
previously described (6). Briefly, Illumina CASAVA (Consensus
Assessment of Sequence and Variation) software (version 1.8) was
used for de-multiplexing and masking of dual index adapter
sequences. Sequence reads were aligned against the human reference
genome (hg19) using NovoAlign with additional realignment of select
regions using the Needleman-Wunsch method (36).
[0233] Candidate somatic mutations, consisting of point mutations,
small insertions, and deletions were identified using VariantDx
(36) across the targeted regions of interest as previously
described (6). Briefly, an alteration was considered a candidate
somatic mutation only when: (i) Three distinct paired reads
contained the mutation in the plasma and the number of distinct
paired reads containing a particular mutation in the plasma was at
least 0.1% of the total distinct read pairs; or (ii) Four distinct
paired reads contained the mutation in the plasma and the number of
distinct paired reads containing a particular mutation in the
plasma was at least 0.05% and less than 0.1% of the total distinct
read pairs; (iii) the mismatched base was not present in >1% of
the reads in a panel of unmatched normal samples as well as not
present in a custom database of common germline variants derived
from dbSNP; (iv) the altered base did not arise from misplaced
genome alignments including paralogous sequences; and (v) the
mutation fell within a protein coding region and was classified as
a missense, nonsense, frameshift, or splice site alteration.
[0234] Candidate alterations were defined as somatic hotspots if
the nucleotide change and amino acid change were identical to an
alteration observed in .gtoreq.20 cancer cases reported in the
COSMIC database. Alterations that were not hotspots were retained
only if either (i) seven or more distinct paired reads contained
the mutation in the plasma and the number of distinct paired reads
containing a particular mutation in the plasma was at least 0.1%
and less than 0.2%, of the total distinct read pairs, or (ii) six
or more distinct paired reads contained the mutation in the plasma
and the number of distinct paired reads containing a particular
mutation in the plasma was at least 0.2% of the total distinct read
pairs. In order to track clonal changes over time, any alteration
identified in at least one blood draw was followed in the remaining
serial timepoints regardless of whether mutant allele fractions fit
the criteria defined to call a single hotspot or non-hotspot
mutation.
[0235] Common germline variants were identified and removed if
present in .gtoreq.25% of reads or <25% of reads if the variant
was recurrent and the majority of alterations at that position had
a mutant allele fraction .gtoreq.25%. Variants known to be at a
somatic hotspot position, or producing a truncating mutation in a
tumor suppressor gene were not excluded as germline changes.
Because of the high frequency of mutations in specific genes and
the possible confounding between somatic and germline changes, we
limited analyses in the APC gene to frameshift or nonsense
mutations, and in KRAS, HRAS and NRAS to positions to 12, 13, 61,
and 146. Finally, we excluded hematopoietic expansion related
variants that have been previously described (28-32), including
those in DNMT3A, IDH1, and IDH2 and specific alterations within
ATM, GNAS, JAK2, or TP53 (Table S3 and Table S6).
[0236] Primary processing of next-generation sequence data for
analyses of copy number alterations in cfDNA samples was performed
as follows: Bam files were preprocessed by successively running
CleanSam and MarkDuplicates from Picard Tools version 2.9.0.
(http://broadinstitute.github.io/picard). Sequence reads were
aligned against the human reference genome (hg19) using
NovoAlign.
[0237] Candidate somatic structural variants were identified
through analyses of low-coverage whole-genome sequencing data
obtained from off-target reads mapping outside of the targeted
capture of 58 cancer driver genes (Table 1 and Table S3) in areas
of the genome farther than 1000 base pairs from the start or end of
a targeted region. Off-target reads were divided into 100 kb bins
with the exception of filtered bins i) with less than 10 kb due to
spacing of target regions, ii) having GC content less than 30% or
greater than 70%, iii) where 25% fell within the ENCODE Duke
Excluded Regions Track
(http://genome.ucsc.edu/cgi-bin/hgFileUi?db=hg19&g=wgEncodeMapability).
The total number of unique reads mapping to each bin were counted
to exclude filtered regions:
s b = log 2 ( 1 0 0 0 0 0 x b - f b r b ) ##EQU00001##
where r.sub.b is the number of unique reads mapped to bin b,
x.sub.b is the length of bin b, and fb is the number of filtered
base pairs within bin b, and the normalized score, s.sub.b, was
assigned to each bin. To remove GC-bias and normalize for
sequencing depth we used LOESS smoothing to predict a bin's
normalized score from the bin-specific GC content. The GC-corrected
score for each bin, , is defined for bin b by subtracting the
predicted score from s.sub.b and exponentiating this using base 2.
We summed the GC-corrected scores for each chromosome arm. The
summed score for a given chromosome arm was divided by the summed
score using all bins to calculate the percentage of genomic
representation.
[0238] Z scores were calculated as previously described (10) for
each chromosome arm for each time point and patient assessed to
determine areas of genome over or under representation. PA scores
were calculated as previously described (10) for each time point
for each patient assessed in order to concisely represent the
aneuploidy observed in each sample by using the five chromosomes
arms with the largest absolute z scores. PA scores higher than the
threshold score of 2.4 provide a specificity greater than 90%
(Student t distribution, three degrees of freedom) for the presence
of aneuploid circulating tumor DNA.
Cell-Free Tumor Load
[0239] Sequence and copy number alterations were detected in cfDNA
for each patient at each time point analyzed and used a tiered
approach to evaluate tumor burden. For patients with detectable
sequence alterations, the mutant allele fraction of the most
abundant alteration in a clone targeted by the TKI was used as
readout of cfTL. In patients without detectable sequence
alterations, we evaluated the PA score as a binary readout of cfTL
where a score above 2.4 indicated aneuploidy and evidence of tumor
burden and a score below 2.4 indicated normal ploidy and the
absence of detectable tumor burden in plasma.
[0240] Changes in cfTL were evaluated to compare tumor burden at
baseline and at other time points during treatment using
quantitative assessment of cfTL mutant allele fractions for
patients with detectable sequence clones and qualitative assessment
of change from aneuploidy to normal ploidy representing a complete
response for patients without detectable sequence clones.
Statistical Analysis
[0241] Significance was determined using a variety of methods. To
assess the significance of reduction in cfTL (FIG. 3A), reduction
in PA scores (FIG. 3B), and change in the number of sequence
mutations detected (FIG. 3C) in patients with radiographic response
or stable disease versus patients with no radiographic response
post treatment we used the Wilcoxon signed rank test. The rates of
emerging mutations in the presence (within 4-12 hours) and absence
of selective pressure of therapy were compared using a
Gamma-Poisson Bayesian model. A Gamma (1, 100) prior was used for
both mutation rates. Reported rates were based on the posterior
mean and 99% posterior credible intervals (CI) (FIG. 4). We
compared progression-free survival in ctDNA responders versus ctDNA
non-responders (FIG. 5C) as well as in RECIST responders versus
RECIST non-responders (FIG. 9) using the Mantel-Cox log-rank test.
Paired t test was used to assess the difference in the time to
response assessment post therapy based on ctDNA analyses versus
RECIST (FIG. 5D).
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OTHER EMBODIMENTS
[0279] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *
References